Glycerol-Free Equilibration with the Addition of Glycerol Shortly before the Freezing Procedure: A Perspective Strategy for Cryopreservation of Wallachian Ram Sperm.

This study investigated the effect of glycerol added in different phases of sperm equilibration on CASA and flow cytometry parameters of thawed ram spermatozoa. Sperm was collected from adult Wallachian rams. The freezing extender was glycerol-free ANDROMED® (Minitub GmbH, Tiefenbach, Germany) supplied by 6% exogenous glycerol at different stages of the cryopreservation process. The purpose of this study was to compare two strategies of glycerol addition for sperm cryopreservation. The first strategy included the use of a glycerol-free extender for the procedure of glycerol-free equilibration and chilling, with the glycerolation of the extender by 6% glycerol shortly before sperm slow freezing (GFA). The second strategy included the use of a freezing extender already glycerolated by 6% glycerol before the equilibration and chilling of sperm and following slow freezing (GA). Sperm samples were analyzed after equilibration (but before freezing) and after thawing (at T0, T1 h, and T2 h time points). iSperm® mCASA (Aidmics Biotechnology Co., LTD., Taipei, Taiwan) was used for the evaluation of sperm kinematics. Flow cytometry was used to measure sperm viability (plasma membrane/acrosome intactness) and mitochondrial membrane potential. The obtained results significantly demonstrated that the glycerol-free equilibration with the addition of glycerol shortly before freezing is a perspective strategy for cryopreservation of Wallachian ram sperm.

Complex Relationships between Milking-Induced Changes in Teat Structures and Their Pre-Milking Dimensions in Holstein Cows.

The study aimed to explore the relationship between teat structure dimensions and their short-term reaction to milking, to find the optimal dimensions of teat structures in relation to milking-induced teat tissue changes. Teat structures (teat length, canal length, thickness at barrel and apex, wall and cistern width) were measured by ultrasonography before and after milking for 38 Holstein cows at the beginning, middle, and end of lactation. We found that milking-induced changes in teat structures significantly depended on their pre-milking size. Furthermore, we observed that some teat structures and their changes were interconnected, and some did not affect each other. For example, changes in the barrel thickness and cistern width were affected by all structures, while the canal and apex did not influence each other. We deduced that more favorable changes were observed for teats of medium length, medium barrel and apex thickness, with teat canals of medium length, but with wider cisterns and thinner walls. The results of this study may help improve research in the area of milking-induced changes in teat morphology. Our findings could help understand potential health risks to animals in relation to teat morphology, milking equipment, and machine settings.

Dynamics of Milk Parameters of Quarter Samples before and after the Dry Period on Czech Farms.

This study aimed to monitor milk parameters on three different dairy farms in the Czech Republic to describe their readiness for implementing selective dry cow therapy. Fat, protein, casein, lactose, solids-not-fat content, total solids content, freezing point, titratable acidity, and somatic cell count of quarter milk samples collected from tested Holstein cows were evaluated. Associations between the tested parameters, as well as the effects of parity, farm, day of calving, and time of evaluation at dry-off and after calving, were assessed. Values of the leading milk components dynamically changed between dry-off and after calving, but only protein content was significantly affected. The most important parameter of our research, the somatic cell count of quarter milk samples, was also not affected by the time of evaluation. Even though a slight increase in the mean of somatic cell count is expected before the dry period and after calving, at dry-off, we observed 30%, 42%, and 24% of quarters with somatic cell counts above 200,000 cells per mL, while after calving, we observed 27%, 16%, and 18% of quarters with somatic cell counts above 200,000 cells per mL on Farm 1, Farm 2, and Farm 3, respectively. High somatic cell counts (>200,000 cells per mL) indicate bacterial infection, as confirmed by the significant negative correlation between this parameter and lactose content. In addition, a deficient milk fat-to-protein ratio was observed on two farms, which may indicate metabolic disorders, as well as the occurrence of intramammary infections. Despite the above, we concluded that according to the thresholds of somatic cell counts for selective dry cow therapy taken from foreign studies, a large part of the udder quarters could be dried off without the administration of antibiotics. However, it is necessary to set up more effective mechanisms for mastitis prevention.

A Pilot Study on the Uptake of Propidium Iodide and YO-PRO-1 Iodide through the Pannexin Channels in Wallachian Frozen-Thawed Ram Spermatozoa.

Propidium iodide (PI) and YO-PRO-1 (YPI) dyes are routinely used to determine sperm viability in many livestock species. It is commonly accepted that these dyes penetrate only sperm cells with damaged plasma membranes. Recently, however, the mechanism of dye uptake unrelated to damaged plasma membranes, but instead related to pannexin channels in dog and stallion sperm cells was demonstrated. This pilot study aimed to evaluate the role of pannexins in the uptake of PI and YPI dyes on Wallachian frozen-thawed ram spermatozoa by flow cytometry using probenecid, a specific inhibitor of pannexin channels. Additionally, the expression of pannexins in Wallachian sperm was evaluated directly (by qRT-PCR). The results demonstrate the active role of pannexin channels in the uptake of PI and YPI dyes on frozen-thawed Wallachian ram sperm. In conclusion, when using the PI or YPI exclusion assay to determine Wallachian frozen-thawed ram sperm viability, the danger of overestimating the number of spermatozoa with the damaged plasma membrane must be considered. The observed breed-specific, and more importantly, individual differences in gene expression as well as in dye uptake indicate the need for further studies.

Optimal time for laparoscopic intrauterine insemination performed on ewes detected in natural heat.

The aim of this study was to optimize the laparoscopic intrauterine insemination (LAI) methodology by testing different time intervals between the natural heat detection and ewe insemination. Three experiments were performed in the breeding conditions of Southern Kazakhstan. Ewes (n = 243) were exposed for one hour to direct contact with the teaser rams (once a day, morning or evening). Ewes expressing behavioral symptoms of heat were detected and inseminated with the use of LAI during 210-1290 min (3.5-21.5 hrs) after heat detection. Reproductive traits of lambing rate (LR) and litter size (LS) were recorded according to the births registered at 137 to 152 days post insemination. Our statistical model showed significance only for the effects of ewe age category and the time interval from heat detection to LAI on the LR attribute. The highest LR (38.8%) was detected in ewes at 2.5-3.5 years of age. Corrected least-square means of LR indicated 18.5 hrs. as an optimal time for LAI of ewes in natural heat. In the present study, the percentage value of lambing rate obtained at 18.5 h interval was 70.7%. Therefore, our study suggested an effective methodology to spread valuable genetic information in the sheep population in the regions of extensive farming where heat cycle synchronization is not usually performed. Importantly, our study is among the first ones that follow the European strategy to eliminate the occurrence of hormones in livestock production and the environment.

Milkability of Holstein cows is significantly affected by the incidence of clinical mastitis for weeks after diagnosis.

The main objective of this research communication was to explore the extent of milkability changes caused by the incidence of clinical mastitis. Our second objective was to investigate if the milkability of cows shortly before mastitis incidence significantly differed in comparison with healthy cows. Milk yield, milking time, average milk flow, partial milk flows and the occurrence of bimodal milk flows were monitored during the first 120 d in milk for all cows that calved on the farm during the experimental period (n = 127). A veterinarian diagnosed 27 cows with clinical mastitis, while the remaining healthy cows served as a control group. The period surrounding the mastitis was monitored in a timeline from 2 weeks before to 4 weeks after the diagnosis. We did not observe any significant differences in milkability between the healthy cows and the cows in the pre-mastitis period, which suggests that monitoring these might not be useful for early mastitis detection. Milk yield and milk flow were significantly decreased in the week of mastitis diagnosis compared to the pre-mastitis period. Milking time was not affected in the week of diagnosis, but was significantly increased in the following week, because milk yield returned to pre-mastitis values, but average milk flow was still significantly decreased. Milk yield was normalized to pre-mastitis values as the first of monitored parameters. Milk flow returned to pre-mastitis values in the fourth week after mastitis diagnosis. Milking time was the only parameter that was significantly affected up to a month after diagnosis. Our results showed that milkability changes caused by mastitis are significant and should not be ignored. The ability to adjust milking settings for cows diagnosed with mastitis could become a useful tool for improving mastitis treatment.

Changes in Fatty Acids Profile, Health Indices, and Physical Characteristics of Organic Eggs from Laying Hens at the Beginning of the First and Second Laying Cycles.

The present study compared the fatty acid profile and some physical parameters of eggs from hens reared according to the organic system at the beginning of the first and second laying cycle. A total of 1080 eggs were analysed at the beginning of the first (from the 28th to 30th week of age) and the second (from the 78th to 80th week of age) laying cycle. It was found that the hen ages influenced the egg weight, egg surface area, yolk proportion, and eggshell colour. Albumen and eggshell proportion, albumen, yolk index, Haugh unit score, and eggshell strength were lower in eggs from older hens compared with those produced from younger layers. Monounsaturated fatty acids were found in higher amounts than saturated fatty acids and polyunsaturated fatty acids in egg yolks of eggs from layers only at the beginning of the second laying cycle. The PUFAn-6/n-3 ratio, saturation, atherogenic, and thrombogenic indices were significantly lower in the egg yolks from older hens compared to younger layers. These findings (regarding the eggs from the older ones) prove that it is practical to utilize them in the organic farming system during a period of two years.

Negative Energy Balance Influences Nutritional Quality of Milk from Czech Fleckvieh Cows due Changes in Proportion of Fatty Acids.

The objective of this study was to evaluate the influence of negative energy balance on fatty acids proportion in the milk of Czech Fleckvieh cows after calving. Milk quality was determined based on fatty acid group proportion. Milk quality was evaluated in relation to selected negative energy balance (NEB) traits: body condition change (DEC) and milk citric acid content (CAC) after calving. Milk samples (n = 992) were collected once per week from 248 Czech Fleckvieh cows during the first month of lactation. Fatty acid content (%) in milk samples was determined and results were grouped as saturated (SFA) (hypercholesterolemic or volatile fatty acids) or unsaturated (UFA) (monounsaturated or polyunsaturated). Our results showed that cows with a deep NEB produce milk that is healthier for human consumption. Cows with a more significant DEC or the highest level of CAC in milk had the lowest proportion of SFA and the highest proportion of UFA (p < 0.01). These cows experienced higher physiological stress after calving; however, they produced milk of higher nutritional quality. Nowadays, we can see preventive efforts to mitigate NEB periods as a result of modern breeding trends regarding vitality, robustness, or longevity.

Effect of Milk Intake, Its Composition, and Fatty Acid Profile Distribution on Live Weight of Suckling Wallachian Lambs until Their Weaning.

This study determined effects of milk production, milk components, or fatty acids (FA) profile on live weight of suckling lambs till their weaning. Live weight (LW, kg) of 42 purebred Wallachian lambs (from 33 ewes) was recorded during four control days with approximately 30-day intervals during rearing. At the same time, their mothers were examined for milk production (kg), milk fat (g), proteins (g), lactose (g), and fatty acids (%) contents. Results investigated using linear regression analysis showed 5.93 kg (p < 0.05) increase of lambs LW corresponded with 1 kg of ewe's daily milk production increase during the observed period. Similarly, significant 0.13 kg or 0.11 kg increases of lambs live weight corresponded with 1 g increase of milk protein or milk lactose at this time. Milk with higher prevalence of trans-palmitoleic acid, trans-vaccenic acid, cis-vaccenic acid, linolelaidic acid, linoleic acid, or conjugated linolenic acid (CLA) significantly improved lambs LW. Moreover, significantly positive Pearson partial correlation between LW and trans-vaccenic acid (r = 0.305) or CLA (r = 0.347) indicated on genetic correlation between these traits. Therefore, milk (natural or artificially supplied) with higher distribution of these specified FAs could improve lambs' LW.

Manipulation of spermatogonial stem cells in livestock species.

We are entering an exciting epoch in livestock biotechnology during which the fundamental approaches (such as transgenesis, spermatozoa cryopreservation and artificial insemination) will be enhanced based on the modern understanding of the biology of spermatogonial stem cells (SSCs) combined with the outstanding recent advances in genomic editing technologies and in vitro cell culture systems. The general aim of this review is to outline comprehensively the promising applications of SSC manipulation that could in the nearest future find practical application in livestock breeding. Here, we will focus on 1) the basics of mammalian SSC biology; 2) the approaches for SSC isolation and purification; 3) the available in vitro systems for the stable expansion of isolated SSCs; 4) a discussion of how the manipulation of SSCs can accelerate livestock transgenesis; 5) a thorough overview of the techniques of SSC transplantation in livestock species (including the preparation of recipients for SSC transplantation, the ultrasonographic-guided SSC transplantation technique in large farm animals, and the perspectives to improve further the SSC transplantation efficiency), and finally, 6) why SSC transplantation is valuable to extend the techniques of spermatozoa cryopreservation and/or artificial insemination. For situations where no reliable data have yet been obtained for a particular livestock species, we will rely on the data obtained from studies conducted in rodents because the knowledge gained from rodent research is translatable to livestock species to a great extent. On the other hand, we will draw special attention to situations where such translation is not possible.

Analysis of fatty acid profile in milk fat of Wallachian sheep during lactation.

In this Research Communication we evaluate the fatty acid (FA) profile of Wallachian sheep milk. The study was performed on 38 ewes in Beskydy Mountains. Samples were collected 4 times during the lactation, at monthly intervals. FA profile as well as groups of saturated, monounsaturated (MUFA), and polyunsaturated (PUFA) FAs were investigated. Considerable increase over the lactation was detected for lauric, myristic, myristoleic and palmitic acids, while stearic acid showed the opposed tendency. Variability, supported by significant differences among particular days of milk collection, was demonstrated for oleic acid; its highest distribution occurred at the beginning and at the end of the trial. The highest distribution of CLA was at the second sample day. Milk of Wallachian sheep naturally grazed at permanent pasture areas showed higher content of PUFA and MUFA in contrast with intensive or semi-intensive sheep breeds reported in the literature.