Lupine Publishers | The Effect of the Application of a Ruminal Activator (VITAFERT) in The First Phase of the Lactation Curve and its Influence on the Bromatological Quality of Milk

 Lupine Publishers | Journal of Dairy & Veterinary Sciences

Abstract

A total of 46 milking cows (animals in production) were selected, with a live weight of 454kg in their third lactation. Production at the beginning of the experiment was 11.3 liters/day, the animals were under the same driving conditions, the main food base was the star grass (Cynodon nlemfuensis) and a load of 2 cows/ha with a rotation period of 35 days. The animals were distributed in two groups by treatment (23 with Norgold and 23 with VITAFERT), each group was formed with animals from 10 to 100 days of lactation distributed in two treatments. Treatment 1: Norgold per cow per day. Treatment 2: 1.65kg of Norgold and 2 liters of VITAFERT (4.4ml/kg body) per cow per day. The objective of this work is 3,4kg to determine the effect of the application of the ruminal activator on the production and bromatological quality of milk. Consumption of VITAFERT significantly improved milk production in dairy cows from 10 to 100 days of lactation. The use of VITAFERT as a ruminal activator increases milk production and some dairy components. The supplement with VITAFERT is an estimated saving of imported (concentrated) foods and improved their use. It is recommended to include THE VITAFERT in the feeding of dairy cows as a way to increase milk production, favoring a partial substitution of the concentrate. Continue long-term studies on the effect of VITAFERT (ruminal activator) on dairy cow production indicators

Keywords: Dairy cows, milk quality, ruminal activator, (VITAFERT)

Introduction

The dairy industry is one of the most dynamic branches in the livestock sector, perhaps due to the increase in demand for milk in the world, stimulated among other causes by the growth of the population on the planet. Another important aspect in the dynamism of this industry is that every time, consumers demand better quality dairy products, which leads to the search for options to try to cover both the demand for milk, its quality and that of its by-products. Currently a series of research work is carried out to find ways to increase concentrations in the main dairy components in a constant way, placing a particular emphasis on fat, protein and lactose; because these components have an effect on both the taste and yield of milk and also on the performance and sensory characteristics of dairy by-products, as well as in the unsaturated fatty acid chain [1]. Tropical pastures and fodder often have particularly low digestibility of dry matter and reduced protein content, limiting microbial activity in rumen and production. These pastures can be used more efficiently when the bacterial populations of the rumen meet their energy requirements, essential protein constituents, minerals and other nutrients [2] as well as, ensuring a level of non-structural carbohydrates, necessary to optimize the synthesis of microbial protein of the ration [3-5].
In Cuba, at the Institute of Animal Science Elías & Herrera [6] reported on the production and use of a new product obtained by a simple biotech process composed of lactobacilli, yeasts, organic acids of short chains and low pH capable of controlling the development of E. coli, significantly reducing the incidence of diarrhea in animals, increasing the gain of live weight and increase the retention of energy and nitrogen. This product is called VITAFERT (MEBA), which has been used in various species with acceptable results. VITAFERT, as part of the concept of efficient activated beneficial microorganisms (MEBA), is a biological product composed of bacteria, yeasts and their metabolites, capable of producing appreciable amounts of short-chain organic acids such as lactic, acetic, propynic, succinic and pyruvic, vitamins and enzymes. It is a fermentation activator that stimulates the production of organic acids, decreases pH, increases and stabilizes the protein, increases the digestibility of dry matter and decreases the cell wall fractions of food materials that undergo its action [6,7]. The objective of this work is to determine the effect of the application of a ruminal activator (VITAFERT) on the production and bromatological quality of cow’s milk.

Materials and Methods

In a typical cow house of the Institute of Animal Science, a total of 46 milking cows (animals in production) were selected, with a living weight of 454kg in their third lactation. Production at the beginning of the experiment was 11.3 liters/day, the animals were under the same driving conditions, the main food base was the star grass (Cynodon nlemfuensis) and a load of 2 cows/ha with a rotation period of 35 days. The animals were divided into two treatments. Treatments consisted of: A.3,4 kg of Norgold per cow per day (300 grams/liter according to initial production, from the first liter). B: 1.65 kg of Norgold (50% less than A) and 2 liters of VITAFERT (4.4ml/kg pv) per cow per day, one litre was offered in each milking. Additional charges were offered during milking hours (4:30 AM and 2:30 PM). The evaluations were carried out over a 45-day period. The animals were properly identified according to treatment. The determinations made consisted of: Milk production per cow (L/cow/day); Bromatological composition of milk per cow (morning and afternoon); Body condition according to the method of 5 points, every 10 days. The animals kept an individual monitoring of the production of milk with weighing at 7, 14, 21, 28, 35, 42 days and sampling of the milk to obtain its bromatological composition, at the same time the body condition of these animals was observed. The bromatological composition was performed in the ICA ruminant laboratory, using milko-Scan 104, A/S N Foss Electric, Denmark. An analysis of processed variance was performed in the Microsoft Excel electronic tab and analyzed through the INFOSTAT statistical package. The economic analysis determined the total revenue from the production of milk per diet supplied, in the period of 45 days, after the experiment, as well as the cost of both diets for an animal in the period analyzed.

Results and Discussion

Table 1 shows the results in the two treatments, significant differences are obtained for milk production, in favor of the treatment with supply of VITAFERT compared to the treatment with Norgold those that may be related to what Elias & Herrera [6] who point out that VITAFERT is a fermentation activator that stimulates the production of organic acids, decreases the pH, increases and stabilizes the protein, increases the digestibility of the matter dries and decreases the cell wall fractions of food that are subjected to its action, Although not determined in this test, Pirela [8] noted that an amino acid deficiency, NH3 and energy reduces microbial protein synthesis, digestibility and nutrient utilization, which negatively influences consumption in this sense coincides From Wind and Palm 2015 and, therefore, on animal production perhaps in this experiment. This product (Vitafer) contains yeasts, which can contribute to improve the efficiency of food utilization, favoring production according to Robinson & Garret [9]; Rivas [10] who propose that yeast, in primipara and multipara cows, improve milk yields between 23 days and 56 days postpartum, as a result of an increase in voluntary consumption of MS and FDN aspect that coincides with what Galina [11]. Castro-Madrigal & Jimeno [12] propose that with the increase in the digestibility of MS, fibrous components (FDN, FDA, hemicelulose) and crude protein, there is greater consumption and a positive effect on milk production.

Table 1: Results in production and body condition in 100-day dairy cows.

When the bromatological composition of milk was analyzed in the two treatments, significant difference in protein, total solids and non-fatty solids was observed. While fat and lactose showed no differences. These results could indicate a better balance in diet nutritions with VITAFERT. This may have been given by the increased concentration of precursors and the effect of the diet coinciding with Stokes, Hernández & Ponce, Hernandez & Armenteros [13-15] and Elijah & Herrera [6] who demonstrate in research that there is strong correlation, between the level of degradable protein in the diet and the effectiveness in the synthesis of bacterial protein and consequently influencing the components of milk (Table 2) [16-20].

Table 2:Assessment of milk bromatology in larger components.

Cost Analysis

Sources considerations

1 litre of VITAFER - 0.11 Cuban pesos Gutierrez 2012
1kg of Norgold - 0.24 Cuban pesos Minagri 2008
Price of sale /litre of milk ? 2.50 Cuban pesos
Table 3 shows that it can be produced with cows of this production level, a saving of more than 30ctvs /cow / day in the feeding system containing VITAFER and when analyzing the system (production and food) can produce an economy of more than 10 pesos/cow/day between one system and another, motivated basically by the increase produced in milk.

Table 3: Economic Response/Treatments.

Conclusion

Consumption of VITAFERT (ruminal activator) improves milk production and some dairy components in dairy cows from 10 to 100 days of lactation. The VITAFER supplement can be a saving of imported (concentrated) foods and better use of them, if their combination is carried out in situations similar to those observed in this work. The results suggest future studies of VITAFERT in complete lactation.

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Lupine Publishers | Effect of Nicarbazin on Incubation Variables of Eggs from Creole Hens of Mexico

 Lupine Publishers | Journal of Dairy & Veterinary Sciences

Introduction

Nicarbazin (NCZ) is an equimolar complex of 4,4’-dinitrocarbanilide and 2-hydroxy-4,6-dimethylpyrimidine [1]. It is an anticoccidial agent used in the poultry industry. Its main function is to prevent multiplication and proliferation of parasites of Eimeria genus in the intestinal tract, avoiding tissue damage, decreased feed consumption, reduced nutrient absorption, dehydration, blood loss, skin depigmentation and susceptibility to other pathogens in birds [2]. Nevertheless, its application has not been entirely beneficial. Ott et al. [1] reported that feeding laying hens with 100 ppm or more of NCZ caused depigmentation of the brown shell and reduced hatchability in approximately 60%. Although the mode of action of NCZ for contraceptive activity is unknown, micrographs of vitelline membranes in mallards showed severe degenerative changes and preliminary in vitro studies indicated that it may increase intracellular calcium levels and early degradation of very low-density lipoproteins that comprise egg yolk [3]. However, the effect of NCZ has not been studied in Creole hens of Mexico. Therefore, the objective of this investigation was to evaluate the effect of NCZ on incubation variables of eggs from that genotype of birds.

Materials and Methods

This study was conducted at the Poultry Experimental Farm of the Chapingo Autonomous University at Texcoco, State of Mexico, Mexico. Located at an altitude of 2,247 m [4]. A total of 1922 eggs were collected from a population of 100 Creole hens of 80 weeks of age. Two treatments were evaluated: T2 (Control), 0.00% nicarbazin, and T1, 0.05% of nicarbazin in feed. Nine hundred and sixty-one eggs from the same population were used per treatment. The collection of eggs for T1 lasted 14 days, followed by a cleaning period of 21 days (feed without nicarbazin) and subsequently collecting eggs for T2. Storage was at an average temperature of 68 °F. The fertilization of the eggs was carried out by artificial insemination. Artificial incubation with a constant temperature of 100 °F and 60% relative humidity (RH) was used until day 18, then live embryos were passed to the hatcher (100 °F and RH: 80%). At 12 and 18 days of incubation eggs were candled to identify clear eggs and eggs with dead embryos, then the embryodiagnostic was carried out. Finally, at 21 days of incubation, non-hatched eggs were separated to conclude the embryodiagnostic. Early dead embryo with no blood (ED), 1-3 days of incubation; early dead embryo with blood (EDB), 4-12 days of incubation [5]; intermediate embryonic death (IED), 13-17 days of incubation; late embryonic death (LED) 18-21 days of incubation; infertile (INFERT); contaminated (CONT) and hatched eggs (HATCH) [6-8], were the categories considered in the embryodiagnostic. The feeding program consisted of a single phase with two diets, one containing 0.05% nicarbazin and one without nicarbazin, both diets containing 16% crude protein; 2,800 kcal ME kg^-1 and 4% Ca Table 1. They were provided with a program of 16 light hours. Water was supplied for free access and feed was restricted to 110 g bird^-1 day^-1.

Table 1: ^{1/Provided per kg of diet: vitamin A, 12,000 IU; vitamin D3, 1,000 IU; vitamin E, 60 IU; vitamin K, 5.0 mg; vitamin B2, 8.0 mg; vitamin B12, 0.030 mg; pantothenic acid, 15 mg; niacin, 50 mg; folic acid, 1.5 mg; choline, 300 mg; biotin, 0.150 mg; thiamine, 3.0 mg. Fe, 50.0 mg; Zn, 110 mg; Mn, 100 mg; Cu, 12.0 mg; Se, 0.3 mg; I, 1.0 mg. 2Sand was used as an inert filler. Statistical analysis Frequency data of categories from embryodiagnostic were compared by the chi-square test for independence with the PROC FREQ contingency tests with α = 0.05, SAS Institute Inc. [12].}

Results

Results of the embryodiagnostic can be observed at Figure 1. The values for the seven categories in the embryodiagnostic were different (P<0.05) between treatments. A higher proportion of the eggs were classified as infertile in T1 (34.5%) comparing to T2 (6.6%). Early embryo mortality (ED + EDB = 54.0%) was also higher in T1 with respect to T2 (ED + EDB = 39.5%), however, ED category contributed with most of the dead embryos in both treatments (45.2 and 31.0% for T1 and T2, respectively). IED category just contributed with a minor proportion of the embryo mortality in T1 (2.0%) and T2 (4.3). LED show a value of 2.8 and 21.6% for T1 and T2, respectively. Contaminated eggs represented 2.5% in T1 and 1.0% in T2. As a result of the values in the previous categories of the embryodiagnostic, hatchability values of 4.2 y 26.9% were obtained for T1 and T2, respectively.

Figure 1: Embryodiagnostic categories in eggs from Creole chicken breeders fed feed without (T2) or with nicarbazin (0.05%) (T1). INFERT: Infertile, ED: Early dead embryo with no blood, EDB: Early dead embryo with blood, IED: intermediate embryonic death, LED: late embryonic death, CONT: contaminated egg, and HATCH: hatched eggs.

Discussion

Even though there are previous reports of the negative effect of nicarbazin on incubation parameters of birds [9], there is not information with respect to birds that are not raised under industrial condition environments, as the Creole chickens of Mexico. Results of the current investigation agree with those of Hughes et al. [10] and Jones et al. [11] who found reduced hatchability in eggs from birds exposed to nicarbazin. As stated by Chapman et al. [12], affection of the integrity of the vitelline membrane is the mechanism by which the coccidiostat affects the correct development of the chick embryo, so that early mortality is expected. On the other hand, given what is known about its mechanism of action [3], the first stages of development are the most negatively affected by nicarbazin. In line with this fact, our results agree with Sherwood et al. [1] who found a 3.8-fold increase in early embryo mortality when White Rock breeder hens where fed nicarbazin. So that, embryos without blood spots or blood vessels (ED category in the current study) were the most abundant in the embryodiagnostic. As previously stated, Creole chickens of Mexico are raised under certain degree of harsh environmental conditions in terms of feed, housing, and potential pathogen agents [13,14], as coccidia. So that it was considered the necessity of evaluate the effect of a coccidiostat on incubation parameters of this poultry genotype. The results obtained indicate that nicarbazin should not be used in the feed of Creole breeder hens, given its negative effect on embryo mortality, specifically on the earliest stages of development [15].

Conclusion

Nicarbazin negatively affects the incubation variables, mainly in the early embryo mortality and hatchability, of Creole hens of Mexico.

Acknowledgements

Matus-Aragón, M.A. and Zárate-Contreras. D, thank to National Council of Science and Technology (CONACyT, Mexico) for the financial support for their graduate’s studies.

Conflict of Interest

The authors declare that there is not financial or conflict of interest in this contribution.

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Lupine Publishers | Characteristic Cellular of the Vaginal Epithelium of Llamas (Lama glama) Captive in the State of Jalisco

 Lupine Publishers | Journal of Dairy & Veterinary Sciences

Abstract

With the objective of contributing data regarding the characteristic cellular forms in vaginal smear of Llamas (Lama glama) and to determine their structural forms in different physiologic states, pregnant, nurslings and you empty, they were carried out vaginal smear of 15 Llamas in captivity condition in the State of Jalisco, Mexico. In the empty Llamas of the 10 to the 30 days post weaning the superficial cells spread to go down from 41% to 7% of presence and the 30 days post weaning the intermediate cells they spread to ascend from 58% to 98% of presence. In the pregnant Llamas in the first month of gestation the naviculars cells were observed in more percentage 86% and the cells flakes in smaller presence 10%, while to the 9° month of gestation increase the presence of cells flakes of 10% to 44% and they diminished the naviculars of 86% to 55%. In the Llamas in nursing of the 10 to the 30 days of nursing the cells flakes spread to ascend from 16% to 54%, the opposite happened to the intermediate cells that spread to go down from 83% to 42% to the 30 days of nursing. The cellular differences in the vaginal epithelium of the Llamas could serve like a tool in the diagnostic of gestation and fertility.

Keywords:Camelids; vaginal cytology; gestation; nursing

Introduction

The principle of the vaginal cytology is based on identifying the type and the percentage of cells in the different stages of the cycle estral, since the hormonal changes of the vagina during the cycle are reflected in the morphology of their cells epitelials[1]. The vaginal cytology is used in most of the species to determine stages of the cycle estral in which is the female, for what is a procedure broadly used in veterinary medicine, [2], and it is used with a lot of effectiveness to determine stadiums of the cycle estral in dogs and cats [3]Stornelli[4] mentions that in the canines with estro 80% of superficial cells is observed, while in the cats with estro 88% of superficial cells is observed [5], while in the necklace Pecarí(Tayassutajacu). Mayor et al.[6] found 60% of superficial cells in estro in the vaginal smear of Alpacas (Vicugnapacos). In a work it found differences in the vaginal epithelium among empty and pregnant Alpacas observing similar cells to other species[7]. The camelids has an unique ovarian cycle, they are induced ovuladores, but their hormonal physiology differs of other ovuladores induced as the rabbit or the cat, [8,9] and they don't present estacionality for photoperiod[10]. The camelids are considered as seasonal reproducers in its natural habitat during the warmest, humid months and of more forage readiness[11]. The vagina of the Llamas measures from 15 to 25cm from the hymen to the cervix and approximately 5 diameter cm [12], these characteristics are necessary to understand its ignorance since it takes us to have a poor yield of breeding, compared with other conventional species[13].  

For such a reason the present study intends to carry out a classification at cellular level in the characteristic ways in vaginal smear of Llamas (Lama glama) captive, and to determine its structural forms in different physiologic states as a tool of diagnose reproductive. 

Materials and Methods

15 females were chosen under captivity conditions belonging to hatcheries peculiar of the state of Jalisco, Mexico, with a corporal condition, weigh and age average of 4-5, 115±8 kg and 4.5±1.5, of which 9 were pregnant, 3 empty and 3 nurslings. For the taking of the samples we use the technique described by Wave [14] where we use a hyssop of soaked sterile cotton with solution of chloride of sodium to 0.9% which 8 cm was introduced inside the vagina carrying out rotational movements against the vaginal wall with the purpose of extracting cells that come off in a natural way rotating the hyssop on a portaobjets, later on noticing with ethylic alcohol of 90°, for the tint that of Giemsa it was preferred and you proceeded that recommended by Lynch [15] where once fixed with alcohol it allows to dry off and it dives for a while in the coloring Giemsa from 15 to 30 minutes, later that washes himself with distilled water and dried off to the environment. The observation to the microscope one carries out with increase of 100X and immersion oil, the opposing cells were classified following the approach of Schutte, [16] in you scale, intermediate, naviculars and superficial.

Results

Figure 1: The following figure shows us the cellular activity from the first month of gestation up to the ninth in the 9 copies.

Figure 2: That shows us the cellular differences in the 3 copies of Llamas revised to the 10, 20 and 30 days search it weans.

As it is appreciated in the Figures 1,2&3 previous to the first month of gestation the naviculars cells were observed in bigger percentage 86% and the flakes cells in smaller presence 10%, the opposite to the 9° month of gestation where it increase the presence of flakes cells of 10% to 44% and they diminished the naviculars of 86% to 55%. One can observe that of the 10 to the 30 days post weaning the superficial cells spread to go down from 41% to 7% of presence and the 30 days post weaning the Intermediate ones they spread to ascend from 58% to 98%, what resembles each other with the cat tames that it not indicates the existence of levels estrogenicos in the one post weaning early, [17]. As it is observed in the previous graph, of the 10 to the 30 days of nursing the cells flakes spread to ascend from 16% to 54% and the opposite happened to the intermediate cells that spread to go down from 83% to 42% to the 30 days.

Figure 3: Where show us the cellular characteristics in the 3 Llamas in nursing, from the 10 days up to the 30.

Discussion and Conclusion

In the pregnant Llamas in the first month of gestation the naviculars cells were observed in bigger percentage 86% and the flakes cells in smaller presence 10%, while to the 9° month of gestation increase the presence of flakes cells of 10% to 44% and they diminished the naviculars of 86% to 55%. In the empty Llamas of the 10 to the 30 days post weaning the superficial cells spread to go down from 41% to 7% of presence. To the 30 days post weaning in the empty Llamas the Cells intermediate spread to ascend from 58% to 98% of presence. as long as in the Llamas in nursing of the 10 to the 30 days of nursing the flakes cells spread to ascend from 16% to 54%. The opposite happened to the intermediate cells that spread to go down from 83% to 42% to the 30 days of nursing. The cellular differences in the vaginal epithelium of the Llamas could serve like a tool in the diagnostic of gestation and fertility.

Conflict of Interest

There are not conflict of interest exists.

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Lupine Publishers | Health Benefits of Camel Milk

 Lupine Publishers | Journal of Dairy & Veterinary Sciences

Abstract

The camel is domesticated in various regions of Asia and Africa, it is considered as a friendly and harmless animal that can survive in hard environmental conditions and provides sources of income to its owners in form of milk, meat, and skin by-products. Camel milk is highly nutritive and possesses some unique characteristics which are being investigated nowadays by many researchers and surprising results revealed that it can be utilized as a natural cure for various life-threatening diseases. This work is aimed to review and highlight the importance of camel milk and its therapeutic properties.

Keywords: Camel; Milk; Health

Mini Review

The Camels also called ship of the desert is one of the most abundant type of animal present in mostly arid zones of Asia and Africa. Since ancient times camels have been domesticated for their products like skin, meat and milk [1]. Milk is the most valuable by product of camel, so it is called as “white gold of desert”. According to some researchers, the camel produces more milk than any other livestock species and its duration of milk production is also longer. The whole lactation periods are comprising of almost 12- 18 months and daily production of milk is almost 3-10kg. The milk of camel consists of a 30 % annual caloric intake of the pastoral community diet [2]. The camel milk is having sharp salty taste with pungent smell and appears as dark white in color. Its taste can vary slightly depending on breed, feeding, health status as well as amount of water consumption by camel. When compared to cow milk which retains its properties for just 2-3 days the camel milk retains its quality for up to 12 days when stored at 2 °C temperature. Moreover, it is also stable for 8-10 hrs. at room temperature. The pH of fresh camel milk is normally neutral but can be tasted slightly acidic if kept for longer period of time [3]. The camel milk has some unique physiological characters like the fat globule diameter is also bigger than that of cow, goat, sheep milk (Figure 1).

Camel milk is very much healthy and is consumed in many regions of the world to cure some diseases as well [4]. It contains many vitamins and also higher amount of zinc so it plays a major role in immune system of body as cells of immune system are sensitive to zinc deficiency [5]. Moreover, some studies have revealed that camel milk also possesses insulin-like properties and can control blood sugar due to hypoglycemic properties in diabetic patients. It can be used as a natural medicine for diabetic patients. Along with that properties this miraculous milk has been also proved by many scientists as a natural medicine for autism and some food allergies [6]. Camel milk naturally consists low lactose as compared to cow milk so according to research, it was suggested that camel milk can be utilized by patients having lactose intolerance issues [7]. An animal study carried out in 2010 revealed that fermented camel milk possess increased number of electrolytes including sodium and potassium and it has therapeutic effects on diarrhea in rats. Thus, it can be decided that fermented camel milk can be consumed food for improving nutritive status of the body and also as therapeutic applications [8]. Naturally camel milk has various enzymes that have immunological as well as antibacterial properties and contains varieties of bacteriocins as shown in Table 1. The main enzyme is lysozyme which attacks common invading pathogens by developing primary immune system of the body. Camel milk is naturally rich in this enzyme. Moreover, it possesses natural lactoferrin which prevents microbial growth within the body and kills germs. The concentration of lactoferrin is much more in camel milk as compared to cow, sheep and goat milk [9].

Figure 1: Comparison of fat globule diameter of different animals’ milk [16].

Table 1: Experimental data on isolation of bacteriocin from camel milk from different countries.

The Lacto-peroxidase enzyme has negative effects on tumor growth and is associated with the iodination of thyroid hormones by acting on thyroid peroxidase enzymes they are naturally present in camel milk. The highest concentration of these enzymes is also having impacts on suppressing metastasis of breast cancer cells [10]. A previous study has revealed that camel milk has effect on oxidative stress in autistic children by increasing the concentration of some antioxidant enzymes like glutathione peroxidase, superoxide dismutase along with myeloperoxidase enzyme when they consumed camel milk for two weeks the levels of these antioxidant enzymes were significantly increased as well as autistic behavior was also improved [11]. More experiments were conducted to evaluate the therapeutics properties of Camel milk in some case reports, invitro as well as in vivo experiments and in some clinical trials also. These studies suggest that camel milk can be utilized to cardiovascular diseases, tuberculosis, hepatitis, autoimmune disorders, rickets and liver cirrhosis [12-16] Although various reviews have been done on properties of milk of different domestic animals the camel milk properties are still lacking till now. Thus, this paper is designed to review accessible evidence on the dietary as well as medicinal worth of camel milk and indorse further study regarding the nutritional and medicinal worth of camel milk built on the data from this literature review.

Conclusion

Camel milk is consisting of important nutrients that are required to keep the body healthy. It contains adequate ratio of antibacterial and antifungal agents that are useful for the prevention of various diseases, like diabetic, cancer, cardiovascular diseases, autism, Rota virus diarrhea, lactose intolerance, autoimmune disorders. It is miraculous milk but lacks plenty of research on its qualities, therefore many people are still unaware of its qualities. Thus, further research should be done at molecular levels to evaluate its qualities and impacts on health.

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Lupine Publishers | Combined on-Farm Analysis of Management Procedures and Vaccination Protocols: The Way to Maximize the Health of the Herd

 Lupine Publishers | Journal of Dairy & Veterinary Sciences

Abstract

During a farm animal’s life, there are a lot of situations that carry stress and induce immunosuppression, increasing the risk of infections and diseases. At the same time, veterinarians and farmers make a big effort (technical and economical) to increase immunity, setting complete vaccination programs. Frequently, this prevention through vaccination fails because administration is carried out during immunosuppression periods. We propose an easy, accurate and understandable strategy based on a complete visit of the farm and interview with the farmer. It is purposed to analyze altogether management procedures on farm, other phenomena and vaccination protocols that occur during the ruminant’s life. After a deep analysis, we suggest changes in the farm’s routine in order to maximize health status of the ruminants, reducing stress and immunosuppression periods, increasing efficacy of vaccines and efficiency of the production system. This strategy includes the analysis of the ruminants’ life, day by day, through a spreadsheet.

Keywords: Advice; cattle; management; ruminants; stress

Introduction

Increase or maximize the health of the ruminants in a herd should be a priority for both farmers and veterinarians. Since long, the use of vaccines in order to directly increase immunity but also reduce amount of pathogens in the herd and reduction of clinical signs in infected animals has been carried out [1-3]. It is also well known that stress factors have a negative impact on the immune system and ruminants submitted to it might suffer from stress-induced immunosuppression during several days [4]. Farm animals are submitted to management, physiological status and environment that can induce stress [4,5]. Even when this management is carried out properly and welfare is excellent in the farm, ruminants can be under 30 immunosuppression for a period of time that ranges from hours to weeks [4,6,7].
This stress may, directly or indirectly, induce a decrease of vaccination efficacy, above all when administration of the vaccine is done during this period of immunosuppression. Ideally, ruminants should not be vaccinated during these immunosuppression periods. On the day by day of a farm, this golden rule is often forgotten, and vaccinations are done under stress or immunosuppression periods. Veterinarians tend to assume that vaccination protocols and management strategies they advise for are correctly done. The reality is that on farm routine brings to a bias in between what is set and what is done. The main objective of this manuscript is to provide a strategy for veterinarians, farmers and advisors in order to identify stress periods and vaccine administrations, evaluate if an increase of health and efficiency can be achieved.

Material and Methods

For this strategy a spreadsheet with several columns and multiple lines (Figure 1) is needed and an interview with the farmer/herd manager that will last 1 to 2 hours will be agreed. It is recommended to have agreed an appointment time before, it is crucial not to be in a hurry and start and finish the interview at once. It is also recommended to have this appointment visiting the farm and the animals, as if it was the first time you are in that farm.
Data collected will be all the stressing management, practices or statuses that occur in a farm from day 0 of life until completing first lactation, including dry off and the second parturition. Most common stress factors must be detailed, and the immunosuppression period attributed. It is also essential to detail all the different feeding and allocation that ruminants will have during the period under study. Finally, all the vaccination protocols will be detailed (product and day of administration -age or day after parturition-). All this data will be in columns and placed in the spreadsheet cell corresponding to the day that is done (Figure 2).

Figure 1: Template scheme used during the audit.

Figure 2: Detailed and completed information needed for analysis between 56 to 84 days of a dairy heifer’s life. PCS: number of calves. BRSV (Bovine Respiratory Syncytial Virus), PI-3: Bovine parainfluenza type 3 virus. BVDV (Bovine viral diarrhoea); IBR (Infectious Bovine Rhinotracheitis); BRD (Bovine Respiratory Disease).

Results and Discussion

The result of this appointment and post office work will be a spreadsheet full of different information that includes nutrition, management, immunosuppression, vaccination and disease. This way to gather information, will allow the advisor to detect, very quickly, unwanted phenomena in a farm, which reduce the health status of the animals. It will be easy to detect failures in the application of vaccinations under periods of stress or immunosuppression, different management too close together in time or too many stressing changes at once (i.e. grouping and change of feeding the same day).
Furthermore, it allows the consultant to suggest changes in the tempos of conducting management practices or suggest changes in the days of vaccination so that they do not coincide with periods of stress and immunosuppression. Thus, maximizing the potential of vaccinations, not only in their ability to generate immunity, but also just when it is desired to generate immunity. As examples, vaccination for respiratory pathogens is strongly recommended at least 2 weeks before transport or grouping and vaccination for BVDV is strongly recommended 3 to 4 weeks before insemination/ matting in order to avoid abortion or persistently infected bovines. Surprisingly, the advisor can sometimes suggest changes that will be better for health status but also for the production costs of the farmer. As an example, reduction on feeding milk replacer to calves without any impact on performance (Figures 3 & 4).

Figure 3: Critical point in the heifer rearing process in a dairy farm. Blue oval shows the stress factors in orange (late dehorning, sudden weaning and grouping) in 15 days and the immunosuppression caused, having a negative impact on the booster of respiratory disease vaccination (in red). Red oval shows the consequence: endemic respiratory disease in this group of animals. PCS: number of calves. BRSV: Bovine respiratory syncytial virus. PI-3: Bovine parainfluenza type 3 virus. BVDV: Bovine viral diarrhoea. IBR: Infectious bovine rhinotracheitis. BRD: Bovine respiratory disease.

Figure 4: Final management and vaccination schedule after analysis of situation described in Figure 3.

Blue oval shows the stress factors in orange (late dehorning, sudden weaning and grouping) in 15 days and the immunosuppression caused, having a negative impact on the booster of respiratory disease vaccination (in red). Red oval shows the consequence: endemic respiratory disease in this group of animals. PCS: number of calves. BRSV: Bovine respiratory syncytial virus. PI-3: Bovine parainfluenza type 3 virus. BVDV: Bovine viral diarrhoea. IBR: Infectious bovine rhinotracheitis. BRD: Bovine respiratory disease.
Changes on management procedures, timing on management and vaccination protocol reduced respiratory disease after grouping. Decisions taken were: Dehorning at 15 days of age, gradual weaning and vaccination protocol started a week earlier. Results were a spare of milk and a 60% decrease of BRD cases. PCS: number of calves. BRSV: Bovine respiratory syncytial virus. PI-3: Bovine parainfluenza type 3 virus. BVDV: Bovine viral diarrhoea. IBR: Infectious Bovine Rhinotracheitis. BRD: Bovine respiratory disease. ML: Milk

Conclusion

Continuous and detailed review of management procedures together with stress factors and vaccination protocols in ruminants’ farms is needed to maximize health status. This audit must be individualized. Results and changes set must be written, analyzed and described in an easy, accurate and understandable way for the farmer.

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Lupine Publishers | Invitation to Use Ozone Therapy in the Veterinary Medicine, Especially in Egyptian Cattle Reproduction

 Lupine Publishers | Journal of Dairy & Veterinary Sciences

Mini Review

The aim of this article is to highlight innovative scientific research that deserves attention about ozone treatment to overcome livestock infertility problems. Reproductive efficiency is essential for sustainable dairy and meat productions and failures in reproduction lead to economic losses. There are many challenges affecting fertility in farm animals. Oxidative stress and microbial infections during reproductive cycle of cattle are important causes of decreasing fertility and is considered an important challenge affecting reproduction. These challenges affect the animal during pregnancy, during or after parturition. The risks of physical damage during the birth process or at failure to release the placenta after parturition are often leading to increasing of microbial infections in the livestock.
In the face of declining fertility in cattle, it can be appreciation and knowledge of factors that interfere with the equilibrium regulating reproduction are the first steps of solve infertility problem. The most common method of uterine treatment is either intrauterine or systemic antibiotic administration. Major challenge facing many producers is finding practical, effective ways to improve reproductive performance. This challenge requires the creation of research ideas characterized by scientific creativity and practical application. Oxidative stress (OS), which resulted from all types of stress, occurs when the balance between reactive oxygen species (ROS) and antioxidant is disrupted. Formation of ROS (in low concentration) is a continuous and normal process of cellular metabolism but in higher concentration, it affects cell membrane integrity and functions, damage DNA, lipid and protein metabolism. Most ROS are formed because of the environmental pollutants. Antioxidants (including vitamins C and E) and antioxidant cofactors (such as selenium, zinc, and copper) can dispose, scavenging, or suppressing the formation of ROS [1].
Ozone therapy (OT) is safe and nontoxic, and therefore should be widely used in the practice of veterinary and human medicine [2]. However, in Egypt, ozone therapy is still inadequately used in veterinary practice. OT with small doses is useful and stimulating, while large doses are harmful. Ozone, a highly water-soluble inorganic molecule, is a gas made of three atoms of oxygen (O) with a cyclic structure [3]. Ozone can be produced by generator which produced through flowing air at a constant flow 51/min. with 150 electrical volte. Because of ozone`s instability and short its half-life (40 min at 20 °C), ozone cannot be stored in tanks [3]. The ozonated olive oil can be stored up to 60 days at 4° till used [4].
As a powerful oxidizer and highly reactive molecule, ozone has strong antibacterial, antiviral and antifungal action [5]. Intrauterine irrigations with ozonated distilled water (400ml) with ozone concentration of 4-5 mg/l are done to provide entire contact with the site of inflammation and to exclude any damage to the mucous membrane in different forms of endometritis. On being ozonated the water via the biluminal catheter is introduced into the uterine cavity and then evacuated via the same catheter. The procedure can be repeated 3 times during one session, which is done once a day [6]. Intrauterine infusion with ozonated olive oil can be used as 50-100ml according to the size of each uterine horn. Moreover, Vaginal irrigations with ozonated saline with the volume up to 1liter and ozone concentration of 6-10 mg/L is to be done daily (8-10 procedures per course) and are to be complemented with applications with ozonated oil (1-2 times a day).
Finally, the ozone product proved to be efficient in improvement of fertility in cattle through local treatment of the postpartum uterine mucosa this is, with the advantage of no milk and meat withdrawal period due to antibiotic residues [7]. In addition, ozone has been found to be more effective in the treatment of endometritis and retained placenta in dairy cows, compared to hormonal and/or antibiotic treatment, with no negative effect on the host regarding residues [8].

Ethics

This article is an original review article based on previously published papers and does not contain new original data.

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Lupine Publishers | Haematology and Serum Biochemistry in the Agouti (Dasyprocta spp.): A Neo-Tropical Rodent with the Potential for Domestication

 Lupine Publishers | Journal of Dairy & Veterinary Sciences

Abstract

This review serves to summarize blood cell and serum biochemical values of the agouti (Dasyprocta spp.). The majority of the researches on the blood and biochemical profiles on the agouti were done in many countries, namely Columbia, Brazil, Peru, Trinidad and Tobago. The literature collected for this review spanned over twenty (20) years from 1997 to 2019. The haematological and biochemical values recorded at various locations were tabulated to give a composite reference range for Dasyprocta spp. The blood cells of the agouti were similar to those found in rodents and human with no differences seen in cell types seen between agoutis of different ages or sexes. The majority of articles showed that there was no difference in blood and biochemical values between ages or sexes. However, there were differences seen in calcium and phosphorus levels of pregnant animals and non-pregnant female animals.

Keywords: Brazil; Peru; Columbia; Trinidad and Tobago; Non-Pregnant; Calcium; Phosphorus

Introduction

The agouti a Neo-tropical rodent of the Dasyproctidae family Emmons & Feer [1]. The agouti is utilized for its meat however captive rearing of these animals throughout the neo-tropics are currently being developed. The agouti practiced scatter hoarding which made it essential for the forest environment Silvius & Fragoso [2]. The agouti is one of six neo-tropical species with the potential for domestication Brown-Uddenberg et al. [3]. Recently biological information about the agouti has been reported in the Caribbean and South America. Observation of the diet, nutrition and digestive anatomy has been done. The agouti was reported to be a frugivorous animal which practices caecotrphy, with a preferred food particle size of 6.25mm x 1.25mm in captivity Garcia et al. [4]; Lall et al. [5]; Dookie et al. [6]. However, recently the agouti was described as being an omnivore where observers recorded the animal consuming eggs and animal matter Jones et al. [7]. Infectious pathogens the agouti can harbour have been reviewed but not no clinical effects have been reported Lall et al. [8]. Endoparasites in the wild population of agouti in Trinidad have been investigated Suepaul, et al. [9], in Brazil Lainson et al. [10]. Endoparasities of captive reared populations in Trinidad have been reported Jones & Garcia [11,12]. Morphological studies of the peripheral blood cells of the agouti (Dasyprocta prymnolopha) in captivity were done. Blood cell types found were erythrocytes, reticulocytes, lymphocytes, eosinophils, neutrophils, monocytes, basophils and platelets. The cellular morphology was similar to other rodents and humans Conde Junior et al. [13]. Therefore, the management and monitoring of the health of the agouti in captivity has become important. The use of reference haematological and biochemical data in Dasyprocta spp. will be a valuable tool in the identification of sub-clinical diseases. The objective of this document was to give a comprehensive summary of haematological and serum biochemical reference values

Haematological analysis of Dasyprocta spp.

Peripheral blood cells of the agoutis in captivity were analysed in Brazil. Results showed that there was no difference in the morphology and morphometry of blood cells between sexes. Erythrocytes had an elliptical form without a nucleus; lymphocytes were spherical with scarce cytoplasm having a dense centralized round nucleus Conde Junior et al. [13]. Monocytes were slightly basophilic having a spherical nucleus with central constriction. Neutrophils and eosinophils were spherical with a polymorphic lobulated nucleus. Basophils were observed to have an abundance of cytoplasmic granules Conde Junior et al. [13]. De Aquinos et al. [14] found that haematocrit values were higher in adults, platelets were superior in young females but there was no difference in white blood cells values between age groups and sexes. In contrast, Andrade et al. (2011) found that there was no significant difference in the blood parameter for age and sex in the agoutis (Dasyprocta fuliginosa) sampled. Further work was done in Brazil on the agouti and results showed that there was no significant difference in blood parameters for age or sex, but lymphocyte values were higher for older animals Ribiero et al. [15]. Haematological reference values for Dasyprocta spp. was summarized using information from published work and presented in Table 1.

Table 1: Reference Haematological values of adult male agoutis (Dasyprocta spp.).

Serum Biochemical analysis of Dasyprocta spp.

Dasyprocta spp. were sampled in Brazil and biochemical analysis found there was no difference in parameters based on age or sex of the animal Ribiero et al. [15,16]. Male agoutis (Dasyprocta leporina) captive reared in Trinidad and Tobago were analysed with similar results as described in Brazil Jones et al. [17]. Nunes et al. [18] examined blood glucose, cholesterol, triglycerides, creatinine and total proteins of adult and young male agoutis in Columbia. Nunes et al. [18] values for glucose, creatinine and total proteins were similar to results obtained in Trinidad and Tobago by Jones et al. [17]. However, serum cholesterol and triglycerides were not recorded for the male agouti in Trinidad and Tobago Jones et al. [17]. Serum biochemistry of agoutis (Dasyprocta prymnolopha) was analysed during pregnancy. Finding showed that protein, globulin, urea, creatinine, alanine aminotransferase, glucose, aspartate aminotransferase, and total bilirubin of the pregnant and non-pregnant animals were not significantly different. Calcium and phosphorus were significantly different for pregnant and non-pregnant animal De Carvalho et al. [19]. However, the level of calcium and phosphorus during the entire pregnancy were constant De Carvalho et al. [19]. Serum biochemistry values are seen in Table 2 which was collected from captive reared and wild populations of Dasyprocta spp. with similar results seen at different locations [20].

Table 2: Reference serum biochemical values of agoutis (Dasyprocta spp.).

Sources: Jones et al. [7]; Ribiero et al. [15]; Nunes et al. [18].

Conclusion

Haematological parameters were similar for different ages and sexes in the Agouti (Dasyprocta spp.). Serum biochemical parameters were also similar for different ages and sexes with the only difference seen in pregnant animals. Calcium and phosphorus were different in pregnant animals as compared to non-pregnant animals but during pregnancy there was no difference seen in the blood calcium and phosphorus levels.

Declaration of Conflict of Interest

The authors declare that there was no conflict of interest when constructing this document

Authors Contributions

KRJ searched the databases for relevant in the construction of the review. KRJ formally wrote the draft document. KRJ and GWG revised the draft manuscript. GWG supervised the entire project.

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Lupine Publishers | Histochemical and Histomorphometrical Studies on the Digital Cushion of Heifers and Dairy Cows with Claw Horn Lesions

 Lupine Publishers | Journal of Dairy & Veterinary Sciences

Abstract

Background: The ruminant claw horn lesions seriously diminish histological structure and functional efficiency of the digital cushion of the dairy cows.

Objective: To investigate histological and histochemical properties of the digital cushion in the dairy cow with claw horn lesions, such as white line disease, sole damage and hemorrhage.

Results: The results showed that digital cushion loses its normal histological structure in claw horn lesions.

Conclusion: Further biochemical, physiological and feeding studies on the experimentally induced claw horn lesions are necessary to understand the nature of the events playing roles involving animal feeding in the development of the lesions, to develop new animal feeding strategies aiming to protect and maintain the normal structure of the digital cushion.”

Keywords:Protein Degradability; Goats; Intensive Feeding; Nitrogen Balance; Digestibility

Introduction

Lameness is one of the most important problems negatively affecting milk yield and animal welfare in modern dairy farming in high-performance animals. The cows with laminitis produce lower amount of milk [1-5]. Moreover, cyclic reproductive changes of these cows slow down [6] or cease because of anoestrus [7] or cystic ovarian disease [8]. Lameness is a major animal welfare problem because that it is a painful condition and causes to behavioral changes of the affected cow. A lame cow tends to display low mobility [9], to spend decreased daily feeding time and dry matter intake [10] and lying behaviour may be indicative [11]. In the dairy cow, claw horn lesions [CHL] constitute 65% of the total lesions causing to lameness [12,13]. Although possible roles of risk factors, such as claw conformation and trimming, animal welfare, management, feeding and housing have been evaluated in detail by previous researchers [14-17], pathophysiology of CHL is still unclear. Previous researchers [18,19] have suggested that CHL is arisen from traumatic lesions of the supportive tissues of the claw. Around the time of calving, increase of hoofase enzyme, oestrogen and relaxin secretions results in loosening and increased mobility of suspensor apparatus of third phalanx [19]. The bovine digital cushion locating under the third phalanx has a complex structure, which major part is constituted of fat tissue. The cushion comprises three cylindrical segments [axial, medial and abaxial segments] of adipose tissue surrounded by a thick connective tissue trabecules, those arranged in parallel to each other [20,21]. The pads of the bovine digital cushion play crucial roles in reducing the body load transferred to surface of sole via absorbing of substantial forces acting within the claw [22]. The biomechanical function of the digital cushion in distributing and attenuation of the load transferred to the base of flexor process of the distal phalanx have been well appreciated by previous researchers [21,23,24]. The aim of the present study was to determine histological and histochemical properties of the digital cushion in the heifers and primiparous, second and third parturitions, and multiparous dairy cows managed under similar feeding, housing and welfare conditions. The foot was chosen from the animals with CHL, such as white line disease, sole damage and haemorrhage, in order to define the histology and histochemical changes in the digital cushions of the animals.

Material and Methods

Animals

This experiment was approved by the Ethical Committee of Experimental Animal Production and Research Centre (2011/023) of Veterinary Faculty of Selçuk University. In the study, the 120-cadaver foot (totally 240 hooves) of Holstein heifers and cows were used as materials. The animals were culled because of moderate to severe CHL and lameness in one or more hooves. The claws of the animals with similar feeding, housing and welfare conditions were subjected to a detailed clinical examination prior to slaughter. Information on the age, number of parturitions, calving history and stage of lactation were and recorded, in addition to the clinical findings. Among the reasons given for culling were insufficient milk yield, lameness and infertility. The animals were divided into 5 groups, each consisted of 6 animals, as
group I: heifers,
group II: primiparous cows,
group III: cows with second parturition,
group IV: cows with third parturition,
group V: multiparous cows with more than three calving.
The first group animals were 20 months old averagely; other groups were between 2.6-6.2-year-old. After slaughtering, all the foot was cleaned, and lesions, such as discoloration, sole ulcer and hemorrhages were recorded, frozen at -20°C and kept until use for histological procedure.

Dissection of the Hooves and Obtaining Tissue Samples

Each foot was thawed. The soft tissues of the sole and heel of each hoof were separated following the horn had been removed, in order to fully expose the axial, middle and abaxial pads of the digital cushion (Figures 1A-B & Figures 2A-C) [18]. Axial pad of the cushion was totally removed, fixed in 10% phosphate buffered (0.1M pH 7.4) formal-saline and used for histological procedures.

Figure 1: The study area, green - places of mass appearance of painted lady butterflies (Vanessa cardui L.) in 2019.

Figure 2: Separating the corium (2A), dissecting pads of the digital cushion (2B) and totally removing the pads (2C).

Histology and Histomorphometry

The tissue samples were subjected to routine histological tissue processing. Briefly, the tissue samples were washed with tap water overnight, dehydrated in ethylene series, cleared in xylene and immersed in paraffin. The sections taken at 6μm were stained as follows; hematoxylin-eosin for investigating general histology and histological changes [25], safranin-O (SO) for histochemical properties of ground substance, alcian blue at pH 2.5 (AB 2.5) for staining acid mucins and mucosubstance, alcian blue at pH 4.0 (AB 4) for demonstrating carboxylated glucose aminoglycans in the ground substance, trichrome stain for Type I collagen fibres (Coll I) and other histological detail, Verhoef’s elastic fibre (EL) stain for EL fibers [26]. The specimens were investigated under light microscope equipped with digital imaging system and digital images were recorded. The images were analyzed with digital image analysis software (BS200 PRO-2005).

Statistical Analysis

The data obtained from the digital image analyses were analyzed statistically with one-way variance analysis (ANOVA) and Tukey’s pair wise comparison tests by using SPSS software 14.01 (Release 14.01 Licence No: 9869264).

Results

Figure 3: CHL incidence and histometrical results of the groups.

Clinical findings of the animals prior to slaughter and results of postmortem gross examination of the foot Welfare and management of the animals included into the study were far from than satisfactory. The animals were housed on the concrete floor, under poor hygiene conditions, and claw trimming was irregular. The animals were mainly fed with corn silage, alfalfa, vetch and hay. Findings of the postmortem examinations of the foot showed that the animals suffered from CHL, such as sole ulcers, hemorrhage and folding, white line disease and sole erosions in one or more hooves. The highest percentage of the claws with CHL was in the multiparous cows, in the group 5. The groups 2 and 1 the group followed it respectively. The lowest lower CHL frequencies were found in the groups 3 and 4, and the frequencies of these groups were quite similar (P>0.05, Figure 3).

Gross Macroscopic Findings of the Cushion Segments

In most of the claws with CHL, segmental structure of the digital cushion was diminished. The digital cushion tissues of the heifers (Group 1) were pale and softer, whereas in the cows with the increasing the parturition number, the tissues gained yellowish colour and relatively more brittle and fragile. The cushion segments were also smaller in the animals of the groups 4 and 5, aged and multiparous cows (Figures 4A & 4B).

Figure 4: Macroscopic appearance of digital cushion tissues of a heifer from the group 1 (4A) and a cow from the group 5 (4B). The cushion pads of the group 5 animal are smaller and yellowish in colour.

Histological Findings

In general, the digital cushion pads of the animals were mainly formed of a loose connective tissue and unilocular adipose tissue. Adipocytes appeared as large cells with unstained cytoplasm in all of the used staining procedures (Figures 5-9). Fibrous connective tissue septa surrounded the loose connective and adipose tissues. There were striking structural differences in the histology of the digital cushion of heifers and calved cows. Loose connective tissue in the heifers, was replaced with adipose tissue in the primiparous cows, and fibrous connective tissue rich in collagen fibres in the third lactation cows. In the heifers and primiparous cows, adipocytes were seen as groups, whereas in the cows of the groups 3, 4 and 5, the cells were observed as individual cells and the cell groups consisting of small numbers of the adipocytes. Adipose tissue percentages of the groups are given in the Table 1. The highest adipose tissue percentage (21.55%) was in the heifers, group 1, and the value was significantly (P<0.001) higher than those of the other groups (Figure 3). The groups 3 and 4 displayed similar (P>0.05) adipose tissue percentages. The multiparous animals (group 5) had the lowest adipose tissue percentage (Figure 3). Type I collagenous fibres were seen as thick undulating bundles of the fibres darkly stained with aniline blue in trichrome stained sections (Figures 5A- 5E).

Figure 5: Collagen fibre bundles (arrows) and adipocytes (asterisks) are seen in the sections from lateral claw cushion of the right forefoot of the heifer from the group 1 (5A), in the medial claw cushion of the left hindfoot of the animal from the group 2 (5B), in the lateral claw cushion of the left forefoot of the animal from the group 3 (5C), medial claw cushion of the left hindfoot of the animal from the group 4 (5D) and lateral claw cushion of the left hindfoot of the animal from the group 5 (5E) are seen. In contrast to a definite decrease in the adipose tissue rate, the connective tissue gradually increased from the group 1 to the group 5. Crossmon’s trichrome. Magnification bar: 100μm.

Figure 6: Sections of the digital cushion stained with AB pH 2.5 from the medial claw of the left forefoot of the animal from the group 1 (6A), the lateral claw of the right forefoot of the animal from the group 2 (6B), the lateral claw of the right hindfoot of the animal from the group 3 (6C) are seen. Staining intensity of the ground substance (arrows) in different sections is quite similar. Asterisks depict the adipocytes. AB pH 2.5 stain. Magnification bar: 100μm.

Figure 7: Sections of the digital cushion stained with AB pH 4 from the medial claw of the left hindfoot of the animal from the group 1 (7A), the medial claw of the left hindfoot of the animal from the group 4 (7B), the medial claw of the left hindfoot of the animal from the group 4 (7C) and the medial claw of the right hindfoot of the animal from the group 5 (7D) are seen. Slight differences in staining intensities of the ground substance (arrows) in different sections are seen. Asterisks depict the adipocytes. AB pH 4 stain. Magnification bar: 100μm.

Figure 8: Sections of the digital cushion stained with safranin O from the lateral claw of the left hindfoot of the animal from the group 1 (8A), the lateral claw of the left hind foot of the animal from the group 2 (8B) and the lateral claw of the right fore foot of the animal from the group 3 (8C) are seen. Staining intensities of the ground substance (arrows) in different sections are quite similar. Asterisk depicts the adipocytes. SO stain. Magnification bar: 100μm.

The heifers had significantly (P<0.001) higher type I collagen fibre percentage (27.82%) than those of the other groups those had quite similar (P>0.05) percentages (Figure 3). In AB pH 2.5 stained specimens, the AB positivity was mainly located in the ground substance around the connective tissue fibres, embedded in-between the adipocyte groups (Figures 6A-6C). The positivity was also stronger around the individual adipocytes and the groups constituted of small numbers of these cells. Percentage of the AB pH 2.5 stained ground substance was highest (2.08%) in the group 3 and group 2 followed it (1.96%), whereas the groups 1, 4 and 5 had quite similar (P>0.05) and significantly (P<0.01) lower values than those of the groups 2 and 3 (Figure 3). AB pH 4.0 mainly stained the similar ground substance regions with AB pH 2.5, around the connective tissue fibres, located around the adipocyte groups (Figures 7A-7D). Results of the histometrical investigations of the AB pH 4.0 stained specimens showed that the group 1 and group 4 had similar (P>0.05), and significantly higher (P<0.01) positivity percentages than those of the groups 2, 3 and 5 (Figure 3). In SO stained specimens, the positivity was very weak (Figures 8A-8C) in all groups. The positivity percentages of the groups were quite similar and there were no significant differences (P>0.05) between the groups (Figure 3). Elastic fibres were mainly found in-between the connective tissue trabecules surrounding adipocyte groups and tunica adventitia of the blood vessels, and seen as brownishblack, branching coarse and gently undulating fine fibres, in the sections stained with Verhoeff’s elastic fibre stain (Figures 9A- 9E). The results of digital image analyses showed that the group 2 had the highest (1.89%) elastic fibre percentage and the value was significantly (P<0.05) higher than those of the other groups. The groups 1and 3, and 4 and 5 had quite similar elastic fibre percentages (P>0.05, Figure 3).

Figure 9: Sections of the digital cushion stained with Verhoef’s elastic fibre stain from the medial claw of the right hindfoot of the animal from the group 1 (9A), the medial claw of the left hindfoot of the animal from the group 2 (9B), the lateral claw of left forefoot of the animal from the group 3 (9C), medial claw of the left hind foot of the animal from the group 4 (9D) and the lateral claw of the left hind foot of the animal from the group 5 (9E) are seen. Elastic fibres (arrows) are mainly located in connective tissue trabecules surrounding the adipocyte groups (asterisks). Verhoef’s elastic fibre stain. Magnification bar: 100μm

Discussion

In the present study, statistically significant (P<0.01) differences were found between the CHL frequencies of the groups. Particularly, high incidences of CHL in the primiparous cows (45.80%, group 2) and multiparous (56.20%) cows (group 5) were striking. The differences possibly have arisen from higher incidence of CHL in more than one or all foot of the animals in these groups. Previous researchers [27,28] have reported that the histological development and functional maturation of the digital cushion are not completed before the age of three, and also incidence of sole lesions, such as sole ulcerations and white line disease are relatively higher in the first lactation because that the cushion tissues is less elastic and rich in saturated fatty acids. In accordance with the previous researchers [27,28] Incidence of CHL was higher in primiparous cows in the present study. The roles of the additional loads and forces exposed at late pregnancy, and structural and biochemical changes in the regional tissues, in addition to trauma of both corium of sole and the cushion tissues in the pathogenesis and occurrence of CHL have been discussed in detail by previous researchers [17-19,29,30]. Moreover, collaborative effects of improper, inadequate feeding and housing practices are mentioned among significant disposing factors. In coordination of anatomic structures of the hoof [bones, joints, ligaments and bursae] leads to CHL, mainly sole ulcers and white line disease. Thus, hoof problems occur more commonly among the cows kept in cubicles or concrete yards or straw yards than in the cows kept in cowsheds. Previous researchers [18,31,32] have reported that lameness incidence increases in multiparous cows with increase of ageing and calving, especially after third lactation, due to increasing the amount of the loose connective tissue while reducing adipose tissue, decreasing body condition score [BCS], thinning of the cushion segments. In the present study, a gradually decline was found in adipose tissue percentages of the groups, with ageing and increasing parturitions. Loosening of suspensor ligament system of the hoof results in increasing in mobility of bones, and this situation disposes the sole corium and sole horn to traumatic lesions, and consequently plays significant roles in the development of CHL, such as sole ulcers, white line disease [19,33]. Loosening of suspensor system of the hoof also increases the sensitivity of the claw to the CHL depending on increase of loads applied to the foot by increase in mammary gland and foetus weights, especially during first lactation [34]. Clinical findings and histometrical results of the hooves and higher CHL incidence of the groups 2 and 5 are consistent with previous findings [18,31,32]. Moreover, higher CHL incidence of the group 5, in which significant shrinkage of the cushion tissue was observed, supports the hypothesis suggesting the decline in load absorbing capacity of the cushion increases risk of injury of sole corium. Thus, environmental factors, such as long time standing on the concrete floor, unsuitable housing conditions and in proper claw trimming are effective in embodiment of the sole trauma and haemorrhages in addition to increase of coffin bone motility in late pregnancy and parturition. Unsuitable housing and hygiene conditions in the animals of the present study were in accordance with the high CHL incidence of the animals.

The digital cushion, which is a specified connective tissue, is constituted of connective tissue cells and intercellular substance [ICS]. The ICS contains collagenous, elastic and reticular fibres embedded in an amorphous ground substance. The amount of the amorphous ground substance in young animals is higher than fibrous elements. With ageing, the amount and rate of fibrous elements increase and the rate reverses in older animals. The digital cushion, a member of supporting system of the ruminant hoof locating in subcutis, is a complex structure, composed of white and yellow adipose tissues. There are significant differences in histological structure and organization of the digital cushion among the ungulate animal species. The digital cushion of the ruminant hoof is closely resembled with the cushion of the elephant’s cushion [18]. Equine digital cushion has relatively different structural features. In the horse, the cushion is composed of small number of elastic fibres and closely packaged interlacing collagenous fibres dispersed in acidic mucous matrix, rich in hyaluronic acid. Large spaces between the cells and fibres occupied with mixoid tissue and islets of fibro cartilage tissue [35,36]. Collagen fibres, especially type I fibres are the dominant fibre type of the connective tissues. Although the collagenous fibres have limited elasticity, their tensile strength is enough to meet the high tensile forces of the foot. In the present study, axial pad of the digital cushion constituted of white, unilocular adipose tissue lobuli surrounded by connective tissue trabecules. Fibro cartilage was not observed in the specimens. In some of the groups, trabecules surrounding adipose tissue lobuli constituted of loose connective tissue and contained less amount of type I collagen fibres, the others were rich in the collagenous fibres. Group 1 contained significantly (p<0.001) higher collagen (27.82%) when compared with the other groups. A gradual decline of type I collagenous fibre percentage was typical in the remaining, older and multiparous groups (groups 3, 4 and 5). Nevertheless, they contained quite similar (P>0.05) type I collagen fibre rates. Gradual reduction of type I collagenous fibre rate with the increase of age and calving might arisen from the structural and functional changes occurred in aged animals and during the late pregnancy and post parturition. It might be predicted that shock absorbing capacity of the digital cushion considerably reduced with declining of collagen fibre rate. In accordance with this prediction, the highest (56.20%) CHL incidence was found in the group 5, which had relatively low rate of type I collagen fibres. Moreover, in accordance with the prediction above, previous researchers [19,33,37], also suggested that loosening of the suspensor system of the hoof during pregnancy and post parturition increases sensitivity of sole corium located between coffin bone and sole to trauma, and consequently playing significant roles in the pathogenesis of the CHL, such as sole ulcers and white line disease.

Although the information on the types and distribution of adipocytes in ruminant digital cushion is limited, the heifer cushion contains lesser amount of the fat cells. The digital cushion of the heifers is a whitish and gelatinous in nature containing high amounts of ground substance and lipid content gradually increases through 2-3 parturitions, and gradually decrease starting from 3 years of age [18,21,32]. Because that white fat tissue is an energy source, many metabolic and hormonal mechanisms, such as pancreatic hormones and glucocorticoids are efficient on this fat tissue. Thus, feeding and other metabolic factors are likely to efficient on both amount and composition of fat of the digital cushion. Moreover, it is well known that the fatty acid composition and amounts of the cow’s digital cushion are seriously affected by metabolic disorders, mainly by ketosis. Similarly, previous researchers [38] emphasized that the topic needs detailed evidences. In the presented study, the digital cushion of the dairy cattle comprised of islets of large unilocular adipocytes. In the digital cushion with low levels of fat tissue, individual adipocytes were frequently seen. The animals in the group 1 contained significantly (P<0.001) higher adipocyte rates (%21.55) and the group 2 it, with a significant decline. However, the group 2 contained significantly (P<0.05) higher fat tissue percentage than those of the groups 3, 4 and 5. Rastani [39] suggested that lipids are mobilized from fat tissues in order to be used in milk synthesis. Lipids in white fat depots in the digital cushion are also consumed. Considering the data obtained in this study, it is possible to assume that white adipose tissue in the ruminant digital cushion also can be used as an energy source in ketosis, which is a condition resulting in negative energy balance [NEB], and thus CHL increase post parturition period. It is well known that fat and elastic fibres of the digital cushion play significant roles in shock absorbing mechanism of the cushion via expanding sideways until their viscoelasticity is limited by stretched fibrous connective tissue septa rich in collagen fibres. Elastic fibres support expansion by flattening their undulations and support reversing to the situation before when the applied force is expired. Significant decreases in both elastic fibre and adipose tissue rates result in loss of elasticity of the digital cushion.

There is limited information on the histochemical properties of ground substance of the cow’s digital cushion, and also histometrical results evaluation of these parameters have not been documented previously. In the present study, the results of the AB pH 2.5, AB pH 4.0 and SO stains showed that ground substance of the cow’s digital cushion was rich in hyaluronic acid (hyaluronan) and other proteoglycans, although there were some differences between the groups. Because that hyaluronic acid is a macromolecule increasing the viscosity of the tissue fluid, it consequently augments resilience of the tissue by binding water molecules. Other proteoglycans stained with SO might support the digital cushion in resisting to pressure, since the proteoglycans also show high water binding affinity and tend to gelation [40]. Although the results of the present study indicated that the ground substance of cow’s digital cushion was rich in hyaluronic acid, the hoof material was obtained from the animals with varying degree of CHL lesions. The authors strongly stress that a detailed comparison should be carried out between the findings of healthy and CHL animals. The elastic fibres, which relatively thinner when compared to the collagen fibres, are able to compensate the forces applied between elasticity limits. Tissues gain elasticity and stress bearing features by their elastic fibre content. Elastic fibres are found in hoof soft tissues as in other elastic tissues. Information on distribution and localization of these fibres in the ruminant hoof soft tissues is insufficient. In a previous histological study [38], elastic fibres were found between the collagen fibre bundles in the digital cushion of African elephant. In this study, elastic fibres were more peculiarly observed in-between the collagenous fibre bundles and in blood vessel walls. Morphometrical analyses evidenced that the group 2 displayed significantly (P<0.05) higher elastic fibre percentage (1.89%) than the other groups. Although there was a declining tendency with increase of the parturition number, the other groups had relatively similar elastic fibre ratios. Besides, striking decline of the elastic fibre percentage was determined in the groups 4 and 5 in concomitant with higher CHL lesion incidence. Although further studies are needed, the results of the present study imply that elastic fibre content of the cow digital cushion is quite limited and might play only small role in the functions of the digital cushion and in the development CHL lesions.

Conclusion

Based on the results, it was concluded that CHL seriously affected both histology and functional efficiency of the digital cushion of dairy cows. Results of the present study, which show strong evidence between histological, histochemical and histometrical findings and CHL lesions are noteworthy. However, the results should be compared with those of the healthy animals in further experiments those are involving experimental models. Further studies necessary to understand the nature of the events playing roles involving animal feeding in the development of the lesions, to develop new animal feeding strategies aiming to protect and maintain the normal structure of the digital cushion, in order to maintain and augment its supportive and protective roles, to reduce incidence of laminitis.

Grant Support

This work was supported by a grant from The Scientific and Technological Research Council of Turkey with project no: TOVAG 112O332.

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