Artificial Insemination Station Research Articles

Animal Welfare Assessment Protocols for Bulls in Artificial Insemination Centers: Requirements, Principles, and Criteria

Animal welfare is a complex subject; as such, it requires a multidimensional approach with the main aim of providing the animals with the "five freedoms". The violations of any one of these freedoms could have an influence on animal wellbeing on different levels. Over the years, many welfare quality protocols were developed in the EU thanks to the Welfare Quality® project. Unfortunately, there is a lack of such summarized information about bull welfare assessment in artificial insemination stations or about how disturbed welfare can be reflected in their productivity. Animal reproduction is the basis for the production of meat and milk; therefore, factors contributing to reduced fertility in bulls are not only indicators of animal welfare but also have implications for human health and the environment. Optimizing the reproductive efficiency of bulls at an early age can help to reduce greenhouse gas emissions. In this review, welfare quality assessment will be evaluated for these production animals using reproduction efficiency as a key area, focusing on stress as a main effect of poor animal welfare and, thereby, reduced fertility. We will address various welfare aspects and possible changes in resources or management to improve outcomes.

Associations between insulin-like factor 3, scrotal circumference and semen characteristics in young Norwegian Red bulls.

With the integration of genomic selection in the cattle artificial insemination (AI) industry, bulls are selected for their semen production capacity and fertility at a younger age than previously. Norwegian Red bull calves selected as candidates to become future AI bulls based on their genomic breeding value are kept in a performance testing station from around the age of 3-12months, allowing for sample collection and analysis of different parameters during their pre- and peripubertal period. Insulin-like factor 3 (INSL3) is a small peptide hormone specifically secreted by the mature Leydig cells of the testes. In the foetus, it induces the first phase of testicular descent and is considered to reflect Leydig cell development during puberty; it could therefore be an interesting early indicator of future semen production capacity. The main objective of our study was to evaluate the relationship between INSL3, scrotal circumference (SC), and semen characteristics. This is the first time INSL3 was measured in the Norwegian Red population. We collected blood samples for analysis of INSL3 from 142 Norwegian Red bulls at the performance testing station and measured their SC on the same day. Altogether, measurements were made at four time points: upon arrival at the performance testing station (quarantine (Q): 2-5months) and later at approximately 6, 9 and 12months of age. Information on season and place of birth were made available from the database of the breeding company Geno, together with data on semen characteristics from the test station and the AI station. The median SCs for age groups Q, 6, 9, and 12 were 15, 21.5, 29, and 34cm, respectively. INSL3 was shown to be positively correlated with SC (R = 0.4) but not with any of the semen characteristics. Similarly, we found no correlation between SC and sperm characteristics from data on ejaculates analysed at the performance testing station and AI station. The mean sperm volume for the 31 selected bulls with at least 10 ejaculates produced in the AI station increased from 2.3ml at the performance testing station to 6.4ml at the AI station. The corresponding increase in mean sperm concentration was from 497 million/ml to 1049 million/ml. We conclude that INSL3 exhibits high inter-individual variability in the Norwegian Red bull population, which cannot be explained by the parameters measured in this study. At present, INSL3 cannot be used as a biomarker of sperm production in this breed.

Investigations on bovine herpesvirus 4 infections in cattle in Bulgaria

Cattle infected with BHV 4 show respiratory symptoms, gastrointestinal and postnatal genital disorders. The virus is a cofactor of bacterial infections. This paper reflects data on the prevalence of BHV 4 infections in several herds, among breeding animals and bulls from artificial insemination stations (AIS). One hundred and twenty six serum samples from bulls were investigated by MVNT and 45 (35.7%) were positive. Epizootiological and clinical studies on the prevalence of BHV 4 infections were limited to bovine farms with suspected infection with this virus. To establish seroconversion to the virus, double serum samples from 264 cattle – 165 native-born cows and 99 imported pregnant heifers were investigated by the microvirus neutralisation test (MVNT) and commercial ELISA for the detection of antibodies against BHV 4. Ninety-nine samples (37.5%) were positive in the MVNT and 110 samples (41.6%) – in ELISA. One out of the seven milk samples from cows with signs of mammary glands involvement was positive in the polymerase chain reaction (PCR) performed to detect the gB and Tk virus genes. Four of the ten whole blood samples from acutely ill animals were positive for BHV 4 DNA by PCR. The results of the present study confirmed that the BHV 4 circulated within the Bulgarian cattle population.

Identification of genomic regions associated with total and progressive sperm motility in Italian Holstein bulls.

Sperm motility is directly related to the ability of sperm to move through the female reproductive tract to reach the ovum. Sperm motility is a complex trait that is influenced by environmental and genetic factors and is associated with male fertility, oocyte penetration rate, and reproductive success of cattle. In this study we carried out a GWAS in Italian Holstein bulls to identify candidate regions and genes associated with variations in progressive and total motility (PM and TM, respectively). After quality control, the final data set consisted of 5,960 records from 949 bulls having semen collected in 10 artificial insemination stations and genotyped at 412,737 SNPs (call rate >95%; minor allele frequency >5%). (Co)variance components were estimated using single trait mixed models, and associations between SNPs and phenotypes were assessed using a genomic BLUP approach. Ten windows that explained the greatest percentage of genetic variance were located on Bos taurus autosomes 1, 2, 4, 6, 7, 23, and 26 for TM and Bos taurus autosomes 1, 2, 4, 6, 8, 16, 23, and 26 for PM. A total of 150 genes for TM and 72 genes for PM were identified within these genomic regions. Gene Ontology enrichment analyses identified significant Gene Ontology terms involved with energy homeostasis, membrane functions, sperm-egg interactions, protection against oxidative stress, olfactory receptors, and immune system. There was significant enrichment of quantitative trait loci for fertility, calving ease, immune response, feed intake, and carcass weight within the candidate windows. These results contribute to understanding the architecture of the genetic control of sperm motility and may aid in the development of strategies to identify subfertile bulls and improve reproductive success.

HISTORICAL RETROSPECTION OF SCIENTIFIC RESEARCH WITH PROBLEMS OF REPRODUCTION IN LIVESTOCK SCIENTISTS OF SCIENTISTS OF THE INSTITUTE OF LIVESTOCK INSTITUTE NAAS (XX CENTURY)

An attempt is made in the historical context to comprehensively assess the contribution of the Institute of Animal Husbandry NAAS in the development of biotechnology, to identify priority developments of scientists of the Department of Biotechnology of Reproduction of Farm Animals as one of the main domestic centers of research on farm animals. Track the evolution of industry basic and applied research as a result of the work of scientists in the scientific field for the needs of livestock practice. During the study period (1932-1999) the institution changed its name several times: Southern Institute of Dairy Science (1930-1934), Ukrainian Research Institute of Cattle (1934-1935), Ukrainian Research Institute of Animal Husbandry (1935-1956) , Research Institute of Animal Husbandry of the Forest-Steppe and Polissya of the Ukrainian SSR (1956–1992), Institute of Animal Husbandry of UAAS (1992–2010), now - Institute of Animal Husbandry of NAAS. The formation of scientific research on the problems of artificial insemination dates back to 1932-1941. In the period of postwar reconstruction (1946-1948), scientific topics are characterized by more in-depth development of topics and experiments. The period of the highest creative activity of scientists in 1949–1990 - the existing and new methods of artificial insemination were improved. With the opening of state breeding stations and artificial insemination stations, the widespread use of this method in the practice of animal husbandry began. IV Smirnov's work was the impetus for the development of methods for freezing and long-term storage of sperm of broodstock. Embryo transplant work has been developed. The embryo has been established to implement the latest advances in farm animal biotechnology and to significantly expand the scope of international scientific contacts. At the beginning of the period of revival of Ukrainian statehood: 1991 - 1999 the issues of animal reproduction theory and further improvement on this basis of technologies for obtaining, cryopreservation and use of gametes and zygotes in artificial insemination and embryo transplantation, as well as development of more efficient biological environments and technical means new technologies.

An Evaluation of Semen Quality of Donor Bulls in the Central Artificial Insemination Station (CAIS), Kundasale, Sri Lanka

Fertility assessment of semen donor bulls used in Artificial Insemination programs is an important step in improving dairy industry in Sri Lanka. A study was conducted to evaluate the fertility of semen donor bulls using semen parameters and respective pregnancy rates. The semen donor bulls (n =21) in the Central Artificial Inseminations Station, Kundasale were selected for the study, and the hypo-osmotic swelling test was performed for both fresh and frozen semen of each bull. Data were analysed using statistical function of Excel software version 2010. Presence of high percentage of hypo-osmotic swelling positive spermatozoa in both fresh and post-thawed frozen semen indicated that all bulls studied were highly fertile. A total of 49255 AIs has been performed in four provinces with an overall pregnancy rate of 28.2%. The highest pregnancy rate of 57.6% was recorded in the Northern Province and the lowest pregnancy rate of 22.5% was recorded in the Central Province. The study revealed that there was no significant difference (p>0.05) in semen quality among individual bulls or among breeds. Al though the semen quality of the bulls was in the acceptable range, the pregnancy rate differed significantly (p<0.05) according to the area. In addition, the overall pregnancy rate was (28.2 %) below the acceptable level. Further studies are required to investigate factors associated with semen quality and the low pregnancy rates observed in some of the areas.

Genetic analysis of semen characteristic traits in Norwegian Red bulls at the artificial insemination center

Compared with cow fertility, genetic analyses of bull fertility are limited and based on relatively few animals. The aim of the present study was to estimate genetic parameters for semen characteristics of Norwegian Red bulls at the artificial insemination (AI) center (Geno AI station, Stange, Norway) and to estimate genetic correlations between some of these traits and andrology traits measured at the performance test station. The data from the AI center consisted of records from 137,919 semen collections from 3,145 bulls with information on semen weight, sperm concentration, motility before and after cryopreservation, motility change during cryopreservation, and number of accepted straws made. Data from the performance test station included 12,522 observations from 3,219 bulls on semen volume, concentration, and motility (%) when fresh and after storing for 24 and 48 h. Genetic parameters were estimated using linear animal repeatability models that included fixed effects of year-month of observation, age of bull, interaction between semen collection number, and interval between collections for all traits and type of diluter for postcryopreservation traits. The random effects included test-day, permanent environmental, and additive genetic effects of the bull. Based on records from the AI center, we found that semen weight, sperm concentration, and number of straws were moderately heritable (0.18-0.20), whereas motility had a lower heritability (0.02-0.08). Heritability of motility (%) was higher after cryopreservation than before. Genetic correlations among the semen characteristics ranged from unfavorable (-0.35) to favorable (0.93), with standard errors ranging from 0.02 to 0.22. Among the most precise genetic correlation estimates, number of straws made from a batch correlated favorably with semen weight (0.62 ± 0.06) and sperm concentration (0.44 ± 0.08), whereas sperm concentration was negatively correlated with weight (-0.33 ± 0.09). The genetic correlation between motility (%) before and after cryopreservation was 0.64 ± 0.14, and motility change during cryopreservation had a strong favorable genetic correlation with motility after cryopreservation (-0.93 ± 0.02). The estimated genetic correlation (standard error) between the traits volume, concentration, and motility when fresh measured at the performance test station and their respective corresponding traits at the AI center were 0.83 (0.05), 0.78 (0.09), and 0.49 (0.31). The final product at the AI center (number of accepted straws) correlated genetically favorably with all semen characteristic traits recorded at the performance test station (ranging from 0.51 to 0.67). Our results show that the andrology testing done at the performance test station is a resource to identify the genetically best bulls for AI production.

Acetyl-CoA Carboxylase Alpha Gene Polymorphism and Its Association with Milk Fatty Acid of Holstein Friesian Using Real-Time PCR Method

Milk fatty acids are largely affected by genetic factors. Acetyl-CoA Carboxylase Alpha (ACACA) gene is one of the important genes in regulation and metabolic function of milk fatty acids in dairy cattle. The objective of this study was to find out the relationship of single nucleotide polymorphism (SNP) ACACA gene with milk fatty acid trait in local dairy cattle. A total of 277 samples of Holstein Friesian (HF) were collected from Indonesian Research Institute for Animal Production (IRIAP), Animal Breeding Center and Forage Feed of Baturraden (ABCFFB), Central Java Province, Animal Husbandry Training Center of Cikole (AHTCC), West Java Province, Singosari Artificial Insemination Station (Singosari AIS), East Java Province, and Lembang Artificial Insemination (Lembang AIS), West Java Province, Indonesia. Genotyping of this SNP marker (g.2203G>T) was analyzed using the real-time Polymerase Chain Reaction (PCR) based on the hybridization TaqMan probe as the method for allelic discrimination. Milk samples were analyzed using Gas Chromatography and Mass Spectrometry (GCMS). The results of this study revealed the GG and GT genotypes. The proportion of the GG genotype frequency (0.88) was higher than the GT genotype (0.11) and the G Allele frequency was shown higher than the T allele in all locations, i.e., 0.942 and 0.08, respectively. The ACACA gene g.2203G>T SNP was significant (p<0.05) for lauric (C12:0) and dodecanoic (C17:1) acids. It was concluded that the ACACA gene g.2203G>T SNP could be useful as a marker selection for milk fatty acid such as lauric and dodecanoic fatty acids.

Efek Lama Penyimpanan Semen Beku Sapi Bali pada Pos Inseminasi Buatan terhadap Membran Plasma, Tudung Akrosom Utuh, dan DNA Spermatozoa

The purpose of this study was to determine the effect of frozen storage time for Bali Bull in artificial insemination station in Samarinda City, East Kalimantan on the quality of motility, viability, velocity, abnormality, plasma membrane integrity (MIn), acrosome integrity (AIn), and DNA damage of spermatozoa. The study design used a completely randomized design (CRD) with 5 treatments (storage time) and 5 replications. Frozen semen of Bali Bull used in 2009 (10 years of storage), 2011 (7 years of storage), 2013 (5 years of storage), 2015 (3 years of storage), and 2017 (1 year of storage). The storage time of frozen semen stored for one to ten years in liquid nitrogen at the artificial insemination station in Kota Samarinda, East Kalimantan was still suitable for use in artificial insemination based on motility quality (44.99±2.40%), viability (55.33±2,60%), velocity (0.050±0.002 mm/sec), abnormality (12.87±1.09%), plasma membrane integrity (58.83 ± 1.86%), acrosome integrity (75.48 ± 1 , 61%), and DNA damage of spermatozoa (1.60 ± 0.21%).

The effectiveness of artificial insemination in horse breeding

As a result of achievements in genetics and in technology of artificial insemination` cattle breeders received a powerful instrument of improving animals productive qualities, the possibilities of selecting stallions with a high genetic potential of productivity were expanded` and the rate of genetic improvement of entire populations was accelerated. Sperm from stallions is taken on an artificial vagina. Evaluation of the quality of sperm is carried out for 3 consecutive days. Insemination of mares is carried out at artificial insemination stations. 10 days after ovulation the mares are checked again with a probing stallion. If the mare for 35–40 days has not shown signs of sexual hunting again then it is followed by rectal test for foal.

Duroc boars have lower progeny mortality and lower fertility than Pietrain boars.

In pig production, Pietrain and Duroc lines are often used as terminal sire lines to produce crossbred slaughter pigs. The objective of this study was to identify the differences in paternal fertility and mortality during the suckling period of crossbred progeny from Pietrain and Duroc terminal sire lines. In total, 87 purebred Duroc boars and 68 purebred Pietrain boars were used as terminal sires to produce 1,823 crossbred Duroc litters (D-litters) and 1,705 crossbred Pietrain litters (P-litters) in two production herds. The sows were crosses between DanBred Landrace and Yorkshire (F1). All boars were kept at the same artificial insemination (AI) station, and all semen doses were produced in the same laboratory. The experiment was balanced according to herd, boars, and time, with approximately 13 sows from each herd mated to each boar within each breed. The results showed higher fertility expressed as litter size at birth in P-litters compared with D-litters led to 0.5 higher total number born (TNB) for P-litters (P = 0.0076). However, piglet mortality including number of stillborn piglets was lower in D-litters compared with P-litters (P < 0.0001), and 5 d after farrowing, the average litter size in P-litters ranged 0.4 below the litter size in D-litters (P < 0.027). At 21 d after birth, mean litter size in P- and D-litters were 14.5 and 14.9 piglets per litter, respectively (P < 0.015). This indicated that Pietrain progenies were weaker than Duroc progenies, and it was concluded that use of Duroc boars as the terminal sire line led to lower piglet mortality. In the two herds, the mean piglet mortality rate including still born piglets ranged from 19.5% to 23.6% and from 17.6% to 19.1% in P- and D-litters, respectively.

Source of variation of hematology and blood biochemical profiles of Holstein Friesian bulls in performance test

Increasing knowledge on blood profiles for bulls residing in an artificial insemination station is needed both for their high economic value and because they represent a homogeneous bovine sub-population with specific characteristics different from other categories of cattle. Therefore, the aims of this study were to calculate descriptive statistics for blood variables routinely collected from Holstein Friesian bulls of genetic merit; to investigate the sources of variation (age, season, and genetic merit) and to calculate the relationship among blood analytes and selection indexes calculated on their daughters. Age affected some erythrocytes series (increasing trend) and white blood cell count (decreasing trend). The seasonal factor influenced significantly mainly urea. Total bilirubin was significantly affected by genetic merit, inversely correlated with the official productive selection index. Hematologic variables were mainly correlated with production index, whereas biochemical variables, such as glucose, urea and proteins, were related to fertility index.

8 Effect of Single Layer Centrifugation with Percoll Plus® of Fresh Bull Semen on Plasma Membrane Stability and Lipid Peroxidation After Cryopreservation

The discontinuous density gradient with Percoll® is routinely used for sperm selection before IVF in bovine in vitro embryo production. However, few studies have been addressed to investigate the use of this colloid in single layer centrifugation (SLC) before sperm freezing. Therefore, this study aimed to verify the effect of SLC with Percoll Plus® (PP; GE Healthcare, Uppsala, Sweden) before freezing on plasma membrane stability (PMS) and lipid peroxidation (LP) of frozen–thawed bull semen. Three Nellore bulls housed at the Artificial Insemination Station were used. The ejaculates, 6 of each bull, were collected by artificial vagina. On the day of each collection, semen was first assessed for sperm motility, concentration, and morphology. Then, the ejaculates were pooled and ~1 billion spermatozoa either diluted [D; 1:2 (v/v)] in freezing extender (without glycerol) or undiluted (UN) was placed on top of a 9-mL column of PP at concentrations of 70% or 90%, resulting in the 70D, 70UD, 90D, and 90UD treatment groups. After centrifugation at 839 × g for 13 min, except for the control group, the supernatant was removed and the pellet of spermatozoa was resuspended in freezing extender (plus glycerol) to a final concentration of 50 × 106 spermatozoa mL−1. Then, 0.5-mL straws were filled, cooled, and frozen. Sperm samples of each group and different days of collection were thawed in a water bath at 37°C for 30 s and diluted at 5 × 106 spermatozoa mL−1 in TALP-polyvinyl alcohol (PVA) plus Hoechst 342 (Sigma Co., St. Louis, MO, USA). Then, samples were stained for PMS with the association of MitoSoxRed (Molecular Probes Inc., Eugene, OR, USA) and YO-PRO-1 (Thermo Fisher Scientific, Hampton, NH, USA) and with C11-BODYPY (Thermo Fisher Scientific) for LP. The sperm samples were analysed by flow cytometer (BD LSR, Fortessa, Becton Dickinson, Mountain View, CA, USA), with the data analysed through use of BD FACSDIVATM software (version 6.1). Analysis of variance and Tukey’s test were used for statistical analysis with P &lt; 0.05 taken as significant. A lower percentage of cells displaying no destabilised membrane was found in 70D (27.6 ± 5.6) compared with the control group (43.1 ± 3.0), but no difference was observed among control group, 70UD (37.2 ± 5.1), 90UD (42.2 ± 7.5), and 90D (32.3 ± 6.5). There was a tendency (P = 0.08) for a higher proportion (P &lt; 0.05) of cells showing LP in control group (48.2 ± 5.7), 70D (48.3 ± 8.5), and 90D (44.43 ± 7.3) compared with the undiluted groups. Thus, spermatozoa selection using SLC with Percoll Plus® did not enhance membrane stability, but sperm LP might be diminished by using SLC with lesser dilution or concentration. It is suggested that lower generation of reactive oxygen species occurred when SLC with PP was used, indicating a possible protective effect on membrane phospholipids. Further studies, including anion superoxide, hydrogen peroxide, and mitochondrial membrane potential could elucidate these findings. This research was supported by FAPESP (grant #2015/20986-3), Tairana AI Station and Master Fertility, Brazil.

10 Evaluation of Frozen Sperm Quality After Single Layer Centrifugation with Percoll Plus® of Fresh Bull Semen

The single layer centrifugation (SLC) with Percoll Plus® (PP; GE Healthcare, Uppsala, Sweden) before freezing is not a common technique used for selection of spermatozoa in bovine. Thus, this study aimed to verify the effect of SLC with PP before freezing on integrity of plasma and acrosome membranes (IPAM), phospholipid translocation (PT), and mitochondrial membrane potential (MMP) of frozen–thawed bull sperm. Three Nellore bulls housed at the Tairana Artificial Insemination Station were used. The ejaculates (6/bull) were collected by artificial vagina and assessed for sperm motility, concentration, and morphology. Then, the sperm were pooled and ~1 billion spermatozoa, either diluted [D; 1:2 (v/v)] in freezing extender (FE; tris, fructose, citric acid, egg yolk and antibiotics, without glycerol) or undiluted (UN), were placed on top of a 9-mL column of PP (in 15-mL centrifuge tubes) at concentrations of 70% or 90%, to form the 70D, 70UD, 90D, and 90UD treatment groups. After centrifugation at 839 × g for 13 min, except for the control (C), the supernatant was discarded and the pellet diluted in FE (plus glycerol) to a final concentration of 50 × 106 spermatozoa mL−1. Afterward, 0.5-mL straws were filled, cooled for 5 h at 4°C, and frozen in a programmable freezer (Digitcool, IMV, L’Aigle, France) following the temperature/time curve: from 4°C to –10°C (5°C min−1), –10°C to –100°C (40°C min−1) and from –100°C to –140°C (20°C min−1), in a total of 8 min, when the straws were plunged into and stored in liquid nitrogen until evaluation. Thawed sperm (at 37°C/30 s) was diluted at 5 × 106 spermatozoa mL−1 in TALP-polyvinyl alcohol (PVA) plus Hoechst 342 (100 μg mL−1; Sigma Co., St. Louis, MO, USA). After that, samples were stained for membrane integrity with the association of fluorescent probes propidium iodide (PI, 50 μg mL−1; Sigma Co.), fluorescein thiocyanate (FITC)-Pisum sativum agglutinin (PSA, 1 mg mL−1; Sigma Co.) and Annexin V-APC (BD Pharmingen, Franklin Lakes, NJ, USA), and with MitoStatus Red (20 nM; BD Pharmingen) and YO-PRO-1 (7.5 μM; Molecular Probes Inc., Eugene, OR, USA) for MMP. Sperm samples were analysed by flow cytometer (BD LSR; Fortessa, Becton Dickinson, Mountain View, CA, USA) and the results expressed as percentage of intact cells or qualitative fluorescence expressed in arbitrary units (AU). Analysis of variance and Tukey’s test were used for statistical analysis with P &lt; 0.05 taken as significant. There were no differences between groups for IPAM (values ranging from 45.9 ± 7.0% to 55.6 ± 8.5%). Similarly, results of PT translocation did not differ among the groups (range from 34.7 ± 7.0% to 47.6 ± 7.0%). However, there was a tendency of increasing MMP (P = 0.10) in 70UD (1789 ± 258 UA), 70D (1776 ± 162.1 UA), and 90UD (1757 ± 133.8 UA) compared with C (1368 ± 267.4 UA) and 90D (1356 ± 145 UA). In conclusion, SLC did not compromise sperm membrane functionality and it seemed to select spermatozoa with higher mitochondrial functional activity when centrifuged at the concentration of 70% and 90D. This research was funded by FAPESP # 2015/20986-3, Tairana Artificial Insemination Station, MasterFertility Ltda, Brazil.

9 Single Layer Centrifugation of Bull Semen Through Percoll Plus® Before Cryopreservation

We previously reported that single layer centrifugation (SLC) with Percoll® (GE Healthcare, Uppsala, Sweden) of fresh bovine semen resulted in improved sperm progressive motility and movement, as evidenced by computer-assisted sperm analyzer (CASA) after freezing-thawing. However, no report has been found in the literature on the use of Percoll Plus® (PP; GE Healthcare), a nontoxic colloid, for the same purpose. Thus, this study aimed to verify the effects of SLC-PP before bull sperm freezing on sperm kinematics after cryopreservation. Ejaculates were collected from 3 Nellore bulls (6 from each) using an artificial vagina. After collection, the semen was assessed and pooled, and then 1 billion spermatozoa either diluted [D; 1:2 (v/v)] in freezing extender (FE, without glycerol) or undiluted (UD) was layered on top of a 9-mL column of PP (in 15-mL centrifuge tubes) at concentrations of 70% or 90% to form the 70D, 70UD, 90D, and 90UD treatment groups. Following centrifugation for 13 min at 839 × g [except for the control (C) group], the supernatant was removed and the sperm pellet diluted to 50 × 106 sperm mL−1 in FE medium plus glycerol. Then, frozen–thawed sperm samples were analysed by CASA (MMC Sperm, St. Petersburg, Russia) for the following parameters: total motility (TM, %), progressive motility (PM, %), curvilinear velocity (VCL, µm−1), straight line velocity (VSL, µm s−1), average path velocity (VAP, µm s−1), amplitude of lateral head displacement (ALH, µm), beat cross frequency (BCF, Hz), linearity (LIN, %), and straightness (STR, %). For statistical analyses, ANOVA and Student-Newman-Keuls test were used. Data are presented as mean ± SEM with P &lt; 0.05 taken as significant. No difference was found among the groups for TM, VSL, BCF, and STR. However, the percentage of PM was higher (P &lt; 0.05) in the SLC-selected sperm samples (values ranging from 42.0 ± 7.0 to 47.4 ± 11.4) than in C (28.8 ± 5.0), and ALH was lower in 70UD (1.6 ± 0.12) and 70D (1.7 ± 0.10) than in C (1.9 ± 0.2). Moreover, 70UD (49.0 ± 1.0), 90UD (50.0 ± 3.0), and 90D (50.0 ± 4.0) displayed higher percentage of LIN (P &lt; 0.05) compared with C (45.0 ± 2.0) and 70D (48.0 ± 3.0). On the other hand, similar results were obtained for VCL (from 126.3 ± 8.0 to 130.0 ± 20.5) and VAP (from 82.7 ± 14.5 to 85.1 ± 6.9) in C, 70UD, and 70D, but these values differed (P &lt; 0.05) from those for VCL in 90UD (104.6 ± 10.3) and 90D (97.2 ± 22.0) as well as for VAP in 90UD (72.2 ± 11.0) and 90D (71.8 ± 9.6). These are the first data demonstrating favourable influences of SLC with 70% Percoll Plus® to select distinct sperm subpopulations as evidenced by enhanced PM, LIN, and ALH. Thus, SLC-PP could optimize the production of frozen bull semen by decreasing the number of sperm per insemination dose, and help to circumvent limitations associated with the poor semen quality sometimes found in bulls of high genetic merit. This research was funded by FAPESP # 2015/20986-3, MasterFertility and Tairana Artificial Insemination Station, Brazil.

The dependence of the growth rate and meat content of young boars on semen parameters and conception rate

Boars have a decisive impact on the progress in pig production, however, there is no recent information about the optimal growth parameters during the rearing period for modern breed later used in artificial insemination (AI) stations. Therefore, the objective of the research was to conduct semen parameter and conception rate analyses on the basis of growth rate and meat content assessments made during the rearing of AI boars of different genotypes. The study was carried out between 2010 and 2014 and included 184 boars in five breed combinations: 46 Polish Large White, 50 Polish Landrace, 27 Pietrain, 36 Duroc×Pietrain and 25 Hampshire×Pietrain. Boars were qualified by daily gains and meat content assessment (between 170 and 210 days of life). A total number of 38 272 ejaculates were examined (semen volume (ml), spermatozoa concentration (×106 ml−1), total number of spermatozoa (×109) and number of insemination doses from one ejaculate (n)). The fertility was determined by the conception rate (%). Semen volume, spermatozoa concentration and conception rate (P<0.01), followed by the total number of spermatozoa and insemination doses (P<0.05) were characterized by the highest variability in relation to breed of boars. The effect of daily gains was reported for spermatozoa concentration, number of insemination doses, conception rate (all P<0.01) and total number of spermatozoa (P<0.05). The peak of growth for spermatozoa concentration, total number of spermatozoa, insemination doses and conception rate was achieved for 800 to 850 g gains. Meat content affected semen volume, number of insemination doses and conception rate (P<0.05). Rearing boars while maintaining daily gains at the 800 to 850 g level and 62.5% to 65% meat content helps AI stations to increase the efficiency and economic profitability, and the number of insemination doses to increase by up to 300 doses/boar within a year. The analyses of growth parameters may help increase the efficiency and economic viability of AI stations.

Effect of genetic and environmental factors on semen productionand quality of Madura cattle in Indonesia

The indigenous Madura cattle semen production and quality were evaluated. Total of 2275 semen collection records from 11 bulls were analyzed to evaluate the genetic potency of semen productions. The genetic evaluation was described as an estimation of bull semen volume, sperm concentration and sperm individual motility. Data were analyzed by HGLM-REML with environmental factors of age, seasons, interval of semen collections and frequency of ejaculations as fixed effect. The result showed that the age and the ejaculation frequency affected to all variables; an interval of semen collections affected to both semen volume and sperm concentrations; and seasons only affected to sperm concentrations. Repeatability estimate of semen volume, sperm concentration and sperm individual motility were 0.376, 0.445 and 0.567 respectively. It can be concluded that the quality of ejaculate was less affected by environmental factors, supported by the good adaptation of indigenous Madura cattle under the local climatic conditions and management in the artificial insemination station.

Genome-wide association study for semen production traits in Holstein-Friesian bulls

Identifying genomic regions, particularly individual genes associated with semen quality traits, may be very important for improving sire fertility via selective breeding. The aim of the study was to estimate (co)variance components and effects of single nucleotide polymorphisms (SNP) from the Illumina BovineSNP50 BeadChip (Illumina, San Diego, CA) on semen production traits and to find candidate genes for these traits. The analyzed data set originates from the Polish Holstein-Friesian dairy cattle population and consists of 1,212 bulls kept at 4 artificial insemination stations. For each bull, 5 semen production traits were collected: sperm concentration, semen volume, number of spermatozoa, motility, and motility score. A multitrait mixed model was used to estimate genetic parameters. The parameters obtained were used to estimate SNP effects for each trait separately by the mixed model, which is used in the Polish direct genomic value project. Additionally, genes located in the vicinity of significant SNP were selected as candidate genes. For motility, 20 genome-wide significant SNP, located on 12 autosomes, were identified. For sperm concentration, we found 7 significant SNP: 3 on chromosome X, and 1 on chromosomes 1, 6, 23, and 24. For semen volume and motility score, 3 and 1 significant SNP were detected, respectively. All these SNP were located on chromosome X. For the number of spermatozoa, 12 significant SNP were observed. Six SNP were located on chromosome X, 3 on chromosome 8, and 1 on chromosomes 2, 7, and 16. This study clearly indicated a key role of the X chromosome in the determination of semen quality and emphasized that including such traits into genetic evaluation should be strongly considered.

Microscopic analysis of MTT stained boar sperm cells

The ability of sperm cells to develop colored formazan by reduction of MTT was used earlier to develop a spectrophotometric assay to determine the viability of sperm cells for several mammalian species. It was the objective of the present study to visualize microscopically the location of the formazan in boar sperm cells. The MTT staining process of boar sperm cells can be divided into a series of morphological events. Incubation of the sperm cells in the presence of MTT resulted after a few min in a diffuse staining of the midpiece of the sperm cells. Upon further incubation the staining of the midpiece became more intense, and gradually the formation of packed formazan granules became more visible. At the same time, a small formazan stained granule appeared medially on the sperm head, which increased in size during further incubation. After incubation for about 1 h the midpiece granules were intensely stained and more clearly distinct as granules, while aggregation of sperm cells occurred. Around 90% of the sperm cells showed these staining events. At the end of the staining the formazan granules have disappeared from both the sperm cells and medium, whereas formazan crystals appeared as thin crystal threads, that became heavily aggregated in the incubation medium. It was concluded that formazan is taken up by lipid droplets in the cytoplasm. Further, the use of the MTT assay to test for sperm viability should be regarded as a qualitative assay, whereas its practical use at artificial insemination (AI) Stations is limited.

SROVNÁNÍ PŘÍPRAVKŮ NA ADSORBCI MYKOTOXINŮ A JEJICH VLIV NA VYBRANÉ UKAZATELE KANČÍHO SPERMATU

Effect of two mycotoxin adsorbents with different mechanisms of action were followed in 180 purebred and terminal combination boars reared in artificial insemination stations. The following parameters of boar sperm quality were investigated: volume in millilitres, concentration and motility, and numbers of pathological spermatozoa. When processing the boar semen, the dilution ration and numbers of AI doses were recorded. Compared were two preparations: in case of Preparation 1 the number of evaluated ejaculates was 1,037 while in case of Preparation 2 altogether 1,109 ejaculates were evaluated. Boars receiving diets with Preparation 1 produced more voluminous ejaculates (by 4.1 ml) and their concentration of spermatozoa was also higher (by 39 thous./ml; P ≤ 0.001). Dilution parameters were better as well and numbers produced of AI doses were also higher. In case of Preparation 2 the motility of spermatozoa was a higher while the numbers of sperms were lower. The obtained results demonstrated that a suitable adsorbent can show a positive effect on both quantitative and qualitative parameters of boar sperm.