Search

Single Layer Centrifugation (SLC) through a low density colloid offers an alternative solution to antibiotic use in boar semen extenders, with lower costs compared to high density colloids. The aim of this study was to explore the reproductive performance of sows when using SLC-prepared semen doses without antibiotics, employing low density Porcicoll to prepare semen doses for artificial insemination in a commercial swine herd in Thailand. Ejaculates were divided into two equal parts to create insemination doses, with each dose containing 3000 × 106 sperm/80 ml for intra-uterine insemination in individual sows. The sows were inseminated twice, with the interval between the two inseminations ranging from 8 to 16 h. The CONTROL group consisted of 206 semen doses treated with antibiotics, prepared for insemination in 103 sows, while the SLC group comprised 194 SLC-prepared semen doses without antibiotics for inseminating 97 sows. Fertility and fecundity traits, including non-return rate, conception rate, farrowing rate, and litter traits (i.e., the total number of piglets born per litter, number of piglets born alive per litter, number of stillborn piglets, and number of mummified fetuses), were compared between groups. Furthermore, data on piglet characteristics, including live-born and stillborn piglets (i.e., the prevalence of stillbirth (yes, no), birth weight, crown-rump length, body mass index (BMI), and ponderal index (PI)), were determined. No significant differences in non-return rate (75.7 % vs. 77.3 %), conception rate (73.8 % vs. 73.2 %), and farrowing rate (71.8 % vs. 73.2 %) were observed between the CONTROL and SLC groups, respectively (P > 0.05). Nevertheless, the total number of piglets born per litter in the SLC group was higher than in the CONTROL group (14.6 ± 0.9 vs. 12.3 ± 0.6, respectively, P = 0.049). Interestingly, the prevalence of stillbirth in the SLC group was lower than in the CONTROL group (6.2 % vs. 11.6 %, respectively, P < 0.001). Moreover, the newborn piglets in the SLC group exhibited higher birth weight and BMI compared to those in the CONTROL group (1.36 ± 0.03 vs. 1.26 ± 0.02 kg, P = 0.005, and 18.3 ± 0.3 vs. 17.3 ± 0.2 kg/m2, P = 0.003). In conclusion, employing sperm doses after SLC through a low density colloid in artificial insemination within a commercial breeding operation did not have a detrimental impact on either fertility or fecundity traits but showed potential benefits in increasing the total number of piglets born per litter. Moreover, improvements were observed in the birth weight and body indexes of piglets, and the percentage of stillbirths was reduced. Our findings introduce new possibilities for antibiotic alternatives in semen extenders to reduce the risk of antimicrobial resistance in the swine industry. Additionally, they provide compelling reproductive outcomes supporting the integration of SLC-prepared semen doses into artificial insemination practices. • Single Layer Centrifugation is an alternative solution to antibiotic use in boar semen extenders. • Farrowing rate did not differ between control and SLC groups. • The total born in the SLC group was higher than in the control group. • The prevalence of stillbirth in the SLC group was lower than in the control group. • Newborn piglets in the SLC group had higher birth weight and BMI than control group. [ABSTRACT FROM AUTHOR]

Since bull fertility may be adversely affected by hot humid conditions, the current increase in global temperature is of concern for future livestock production. Heat stress occurs when the body's normal physiological mechanisms to regulate body temperature cannot cope with external conditions. The testes and scrotum have their own complex regulatory mechanisms to protect developing sperm during their most vulnerable stages, but even these may be overwhelmed by unfavourable external conditions. The effects of mild, moderate and severe heat stress are somewhat different, with cattle exposed to mild and moderate heat stress apparently showing an adverse effect on fertility, whereas cattle in very hot, humid climates almost continuously may not exhibit any difference in sperm quality throughout the year. This apparent paradox may be due to differences in the cattle populations being studied, since they could differ in breed, age, purpose (beef versus dairy), or even in the methods used to assess sperm quality. The adverse effects on fertility may occur through the effects of reactive oxygen species on sperm DNA, or through perturbation of the production of antioxidants that usually protect sperm from oxidative attack. These effects can be mitigated to some extent by choosing breed and age of bulls with care, and adopting breeding strategies that avoid semen collection or ejaculation at the most adverse times of year. Husbandry measures such as controlled ventilation, misting, provision of shade or cool surfaces for lying down, could aid temperature regulation. Avoiding heat stress during late pregnancy aids calf growth in early life; careful feeding regimens for young bull calves create good conditions for sperm quality after puberty. Bull fertility is too important to be left to chance. Breeds should be chosen according to climate conditions and the purpose of livestock production. • Bull fertility may be adversely affected by hot humid conditions. • Manifestation of heat stress represents a complex interaction of factors. • Effects of natural mild, moderate and severe heat stress differ. • Scrotal insulation is not a representative model for climate change. • Heat stress can be mitigated to some extent by management and husbandry. [ABSTRACT FROM AUTHOR]

Freezing and thawing processes induce structural and functional damage to sperm plasma membranes and internal organelles. Adding seminal plasma (SP) has been found to minimize or repair the cryoinjuries in some species. The objective of this study was to investigate whether adding SP from stallions of known freezability after thawing could repair cryoinjuries. Semen was collected from warmblood stallions (n = 8, three ejaculates/stallion) and processed by Single Layer Centrifugation (SLC) to remove SP prior to freezing. Pooled SP (5%) from bad freezer (BF) or good freezer (GF) stallions was added after thawing. Post-thaw sperm quality was assessed by flow cytometry in terms of chromatin integrity (%DFI), membrane integrity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and MitoSOX. Sperm kinematics were also assessed by computer-assisted sperm analysis. The %DFI was lower in SLC control (C) than in BF or GF (P < 0.0001, P < 0.0003 respectively). The proportion of viable spermatozoa with intact cell membranes was higher in C than in SP treated groups (C vs. BF, P = 0.02; C vs GF, P = 0.05). There were fewer spermatozoa with low MMP and more with high MMP for C than GF (P = 0.006). The spermatozoa treated with SP from good freezers produced more ROS than when treated with SP from bad freezers (P = 0.007). Motility parameters were not affected by adding SP. In conclusion, adding SP after thawing does not have a beneficial effect on sperm quality, suggesting an inability to repair stallion sperm cryoinjuries, regardless of whether the SP originated from stallions semen, which has good or bad quality after thawing. [ABSTRACT FROM AUTHOR]

There are stallions with apparently good sperm quality that achieve low pregnancy rates when their semen is used for artificial insemination (AI). Thus, additional methods of evaluating sperm quality that result in better methods of predicting stallion fertility, are essential. Semen samples were obtained in April-June from 8 stallions, 4 trotters and 4 warmblood sport horses, which were sampled Monday, Wednesday and Friday each week. From each stallion, 1-4 samples were obtained, and evaluated after cold storage for 24 hours. The samples were evaluated for kinematic variables using CASA, and for membrane integrity, mitochondrial membrane potential (MMP), production of reactive oxygen species, acrosome integrity and chromatin integrity using flow cytometry. Relationships with fertility, calculated as number of pregnant mares/number of inseminated mares for samples used for artificial insemination after transport in cold storage, were evaluated by linear regression, following log-transformation of variables found not to be normally distributed. The following sperm variables were found to have a positive relationship with fertility: Straightness (r2=0.86, p= 0.0009), Viable with High mitosoxred/Low MMP (r2=0.70, p= 0.01), Viable non acrosome reacted (r2=0.68, p= 0.01), Viable by calceinviolet (r2=0.67, p= 0.01), Viable non H 2 O 2 -producing (r2=0.66, p= 0.01), Viable with High mitosoxred/High MMP (r2=0.65, p= 0.02), Viable non superoxide-producing (r2=0.60, p= 0.02), Linearity (r2=0.56, p= 0.03). The only variable found to have a negative relationship with fertility was Amplitude of lateral head displacement (r2=0.63, p= 0.02). The other 26 measured variables did not show a significant relationship with fertility. The results of the study shows that both kinematic variables as well as flow cytometric analysis can be valuable for relating fertility with sperm quality in vitro. The flow cytometry results indicate that analysis of the production of different reactive oxygen species can be valuable when predicting stallion fertility. Acknowledgments: Special thanks to Menhammar, Västerbo, Markebäck and Stockholm Seminstation for providing stallion semen. This work was supported by funding from FORMAS (2018-01083; awarded to AJ). The work was also supported by the Swedish University of Agricultural Sciences via the Cells for Life Platform. [ABSTRACT FROM AUTHOR]

Cryopreservation of epididymal spermatozoa is the last method for preserving gametes of valuable stallions in case of sudden death, severe injuries, or orchiectomy. Due to harvesting methods, sperm is unavoidably contaminated with epithelial and red blood cells, leukocytes, debris, and bacteria. These contaminants and dead spermatozoa can cause detrimental effects on sperm quality. Colloid centrifugation can reduce the load of these contaminants and select a subpopulation of motile spermatozoa. The objective of this experiment was to evaluate whether single layer centrifugation (SLC) prior to cryopreservation or post-thawing improves the quality of stallion epididymal sperm. Ten stallions were submitted to bilateral orchiectomy and harvesting of epididymal cauda spermatozoa (n=20) was performed by retrograde flushing with an extender. Epididymal sperm samples were subjected to conventional centrifugation (no colloid - 600xg for 20 minutes), and SLC prior to cryopreservation (SLC-PC) or SLC post-thaw (SLC+). SLC was performed at 300 x g for 10 minutes (Androcoll-Equine, Minitube, Tiefenbach, Germany). All samples were cryopreserved, thawed, and evaluated. The post-thaw group (SLC+) was thawed, centrifuged by SLC, and resuspended in freezing extender (SLC+F) or cooling extender (SLC+C). Total motility and progressive motility were evaluated with computer-assisted semen analyses (CASA). Sperm morphology was evaluated by examining a wetmount preparation of unstained samples fixed in formol saline. Mitochondrial functionality, membrane integrity, and DNA integrity were evaluated in a fluorescence microscope with specific fluorescent dyes. All variables were normally distributed according to the Shapiro–Wilk test. Data were evaluated by descriptive statistics, one-way analysis of variance, and Tukey's test. Significance was set at p <0.05. SLC-PC (24.6 ± 3.6%) and SLC+F (26.2 ± 4%) treatments yielded significantly higher total sperm motility than SLC+C (16.7 ± 1.7%) and conventional (16 ± 3.5%) treatment. SLC+F (17.5 ± 3.8%) yielded significantly higher progressive motility than the conventional (8.8 ± 2%), SLC+C (9.8 ± 1.3%), and SLC-PC (12.9 ± 4.1%) treatments. The mitochondrial functionality of all SLC groups was significantly higher (SLC-PC 72 ± 2%; SLC+C 74.6 ± 1.8%; SLC+F 82.5 ± 1.9%), than that of the conventional group (25.2 ± 2.1%). Plasma membrane integrity was higher in SLC-PC (48.6 ± 4%) than in SLC+F (30 ± 6.5%), SLC+C (36 ± 4.8%), and conventional (22.8 ± 4.5%) samples. DNA integrity and percentage of morphologically normal samples were unaffected by treatment. SLC is a simple procedure that can be performed prior to cryopreservation or post-thawing and that can improve stallion epididymal sperm quality. Epididymal spermatozoa submitted to SLC only after thawing is best extended using freezing extender than cooling extender. [ABSTRACT FROM AUTHOR]

Antibiotics are added to semen extenders to inhibit bacterial growth and maintain sperm quality in semen doses during storage and transportation. The low concentration of antibiotics used, however, could contribute to increased antimicrobial resistance. Recent studies with boar semen suggest that centrifugation through a low density colloid could separate the spermatozoa from most of the bacteria. Such methods can only be recommended for stallion semen if they are without detrimental effects on sperm characteristics. Therefore, the purpose of this pilot study was to investigate if semen processing by single layer centrifugation through a low density colloid reduces the bacterial load of stallion semen without impairing sperm characteristics. Six healthy and fertile stallions were available at the Centre for Artificial Insemination of the University of Veterinary Medicine Vienna. One ejaculate from each stallion was collected and divided into three parts. Each part was either extended to 15 mL (100 million/mL) and processed by single layer centrifugation with 15 mL of 25% low density colloid (SLC), extended with Equiplus without antibiotics (Minitube, Tiefenbach, Germany), or kept as unprocessed control (raw semen). Aliquots from all samples were sent to the Institute of Microbiology for bacteriologicalexamination where they were serially diluted, plated onto blood agar plates, and incubated under aerobic and anaerobic conditions. The number of bacterial colony forming units (cfu/mL) was determined after 4 days of incubation. Sperm motility and viability in SLC and extended samples were evaluated by computer assisted sperm analysis (CASA; SpermVision, Minitube). The SLC and extended samples were stored at 5°C and re-evaluated after 24 h. For statistical analysis, the mean number of bacterial cfus and sperm characteristics were compared among treatments by one-way ANOVA and Tukey's test, with significance set at p < 0.05. Overall sperm yield after SLC was 76.6% (±16.4). Bacterial cfus were lower in SLC samples and extended samples compared to raw semen (4.5 × 106 cfu/mL, (14.5 × 106cfu/mL, and 8.3 × 106, respectively; p= 0.017, and p = 0.038, respectively). Sperm motility was higher in SLC samples at both time points (93.8% at 0 h, p = 0.001; 87.7% at 24h, p = 0.007) than in extended semen at 24 h (56.3%). Sperm viability was higher in the SLC at both time points (88.4% at 0 h, p = 0.0002; 86.4% at 24h, p = 0.002) than in extended semen at 24 h (73.2%). In conclusion, bacterial load, and sperm characteristics, were improved in SLC compared to extended stallion semen. An extended investigation is warranted. [ABSTRACT FROM AUTHOR]

Traumatic injuries and sudden death can prematurely end the breeding career of a stallion. In such cases, a final spermatozoa collection can be obtained by harvesting the cauda epididymis. Semen samples can then be used for fresh artificial insemination or cryopreserved. Centrifugation is a critical point of sperm cryopreservation processing and can be detrimental to spermatozoa. Colloid centrifugation approaches reduce this physical damage and can be used to select better quality sperm. The aim of this research was to determine the effect of cushioned and single layer centrifugation (SLC) on epididymal stallion sperm motility postcentrifugation. Eight stallions were submitted to bilateral orchiectomy and the resulting epididymal cauda (n = 16) were flushed with semen extender. After harvesting, the samples were submitted to three centrifugation protocols: conventional (20 minutes at 600× g ), cushioned (20 minutes at 900× g ), and SLC (20 minutes at 300× g ). The pellets were resuspended and sperm parameters were evaluated. Sperm morphology was evaluated under a phase-contrast microscope, total motility (TM) and progressive motility (PM) were evaluated with computer-assisted semen analyses. The proportion of morphologically normal spermatozoa was 72.2% for SLC, 72% for cushioned, and 67% for conventional ( P > .05). After conventional centrifugation, it was recorded a TM of 7.4% and PM of 2.7%. After cushioned centrifugation, it was recorded a TM of 13.9% and PM of 6.5%. After SLC, it was recorded a TM of 46% and PM of 32.1%. The motility of spermatozoa recovered by SLC ( P < .05) and cushioned centrifugation ( P > .05) were higher than those recovered by conventional centrifugation. Colloids, including cushioned and SLC, improved epididymal stallion sperm motility postcentrifugation. [ABSTRACT FROM AUTHOR]

The accurate prediction of bull fertility is of major economic importance in the dairy breeding industry. Sperm fertilizing potential is determined by their ability to reach the oocyte, complete fertilization, and sustain embryogenesis, which is partly determined by the quality of sperm DNA. In the present study, we analyzed several sperm functions required for fertility, including DNA damage, in frozen-thawed spermatozoa of breeding bulls with different adjusted nonreturn rates (NRR56), and identified a suitable combination of parameters that could be used to predict bull fertility. Based on the NRR56, bulls were classified into below- and above-average fertility, a total of 37 characteristics of spermatozoa were evaluated for each bull, and their relationship with bull fertility was studied. Of the different sperm functional attributes, differences were observed in sperm viability, acrosomal integrity, reactive oxygen species, and DNA fragmentation index (%DFI) among below-average, average, and above-average fertility bulls. Principal component analysis also revealed that sperm viability, acrosome status, reactive oxygen species, and %DFI were the important variables, having highest correlation with NRR56. Our results indicated that the proportion of live [correlation coefficient (r) = 0.53] and live acrosome-reacted spermatozoa (r = 0.50) were significantly positively related to NRR56, whereas the proportion of dead spermatozoa (r = -0.53) and %DFI (r = 0.61) were significantly negatively related to NRR56 in bulls. Linear regression analysis indicated that a combination of live [coefficient of determination (R²) = 0.72], dead (R² = 0.72), live hydrogen peroxidenegative spermatozoa (R² = 0.64), and %DFI (R² = 0.56) could differentiate below-average and aboveaverage fertility bulls, and thus were considered for development of a fertility prediction model. The accuracy of the developed model for fertility prediction in bulls was high (R² = 0.83). We concluded that flow cytometric detection of sperm viability, hydrogen peroxide status, and %DFI could discriminate below- from above-average fertility bulls. [ABSTRACT FROM AUTHOR]

• Serum insulin-like factor 3 in pre- and peripubertal Norwegian Red bulls was investigated. • Insulin-like factor 3 exhibited high inter-individual variance unrelated to season or geographical birth area. • Scrotal circumference increased from 2-5 to 12 months of age. • There was a moderate positive correlation (0.4) between insulin-like factor 3 levels and scrotal circumference. • Insulin-like factor 3 alone appeared not to be suitable as a biomarker of sperm production onset in this breed. 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 3–12 months, 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) : 2–5 months) and later at approximately 6, 9 and 12 months 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 34 cm, 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 2.3 ml at the performance testing station to 6.4 ml at the AI station. The corresponding increase in mean sperm concentration was from 497 million/ml to 1 049 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. [ABSTRACT FROM AUTHOR]

The present study aimed to evaluate the effect of single-layer centrifugation (SLC) through a species-specific colloid (Androcoll-B; patent pending, J. M. Morrell) on bull sperm quality. Computer-assisted sperm analysis of motility and flow cytometric analysis of sperm viability (SYBR-14/propidium iodide staining), chromatin integrity (acridine orange staining), reactive oxygen species production [Hoechst 33258-hydroethidine-2',7'-dichlorodihydrofluorescein diacetate (HO-HE-DCFDA) staining], mitochondrial membrane potential (staining with JC-1 probe), and protein tyrosine phosphorylation (specific antibody staining) were performed on un-selected and SLC-selected sperm samples. Single-layer centrifugation of bull spermatozoa resulted in the selection of a sperm population that had high mitochondrial membrane potential, a higher content of phosphorylated protein, and more reactive oxygen species than control samples. Sperm chromatin damage was lower in the SLC samples although sperm viability and motility did not differ between SLC samples and controls. These observations suggest that SLC of bull semen in a soybean-containing extender improved some, but not all, parameters of sperm quality. [ABSTRACT FROM AUTHOR]

Abstract: Although several selection techniques are available for processing spermatozoa, only colloid centrifugation has been used to any extent in this field, starting with density gradient centrifugation and progressing more recently to single-layer centrifugation (SLC). SLC through a species-specific colloid has been shown to be effective in selecting spermatozoa with good motility and normal morphology from stallion semen. The method is easier to use and less time-consuming than density gradient centrifugation, and has been scaled-up to allow whole ejaculates to be processed in a practical manner. The potential applications of SLC in equine breeding are as follows: to improve sperm quality in artificial insemination doses for “problem” ejaculates, to increase the shelf life of normal sperm doses, to remove pathogens (viruses, bacteria), to improve cryosurvival by removing dead and dying spermatozoa before freezing or after thawing, to select spermatozoa for intracytoplasmic sperm injection, and to aid conservation breeding. [Copyright &y& Elsevier]

Abstract: Improved sperm selection techniques are needed to increase the efficiency of equine-assisted reproduction. Single layer centrifugation (SLC) of spermatozoa has been shown to improve the quality of stallion sperm samples. In this study, the functionality of selected stallion spermatozoa was tested by intracytoplasmic sperm injection of equine oocytes after selection by SLC through Androcoll-E or by discontinuous density gradient centrifugation (DGC) through Redigrad and Tyrode''s medium with added albumin, lactate, and pyruvate. The mean cleavage rates of the injected oocytes from SLC- and DGC-selected spermatozoa were 67% and 66%, respectively, whereas the proportion of blastocysts developing from cleaved oocytes was 28% and 22%, respectively (P > .05, not significant). An incidental finding was that there was a tendency for SLC-selected spermatozoa to have a higher percentage of spermatozoa with normal morphology than DGC (70% ± 22% vs. 58% ± 38%) and for more blastocysts to be obtained from subfertile ejaculates (21 [19.6%] vs. 15 [14.4%], respectively). In further experiments, stallion spermatozoa bound to hyaluronan, although binding may depend on the semen extender and sperm treatment as well as incubation time. In conclusion, SLC-selected stallion spermatozoa function normally when injected into oocytes. SLC may potentially be better than DGC at selecting spermatozoa from subfertile ejaculates, but this effect needs rigorous investigation with a much larger sample size. Use of the hyaluronan-binding assay for assessing the potential fertility of stallion spermatozoa may be useful but requires further evaluation. [Copyright &y& Elsevier]

Abstract: This study was conducted in an attempt to see whether single-layer centrifugation (SLC) increases the susceptibility of stallion spermatozoa to lipid peroxidation (LPO), in different extenders after removing all seminal plasma (SP). The susceptibility of stallion spermatozoa to LPO was studied before and after SLC. Each ejaculate was split, and aliquots extended with one of the three different extenders: INRA 96, Kenney''s, or Equipro, and stored for 24 hours at 5°C (i). From the extended samples, an aliquot was kept as a control and the other was subjected to SLC through Androcoll-E. The selected spermatozoa were re-suspended in the appropriate extenders, without (ii) or with (iii) addition of 50% (v/v) pooled homologous SP for 24 hours at 5°C. Using ferrous sulfate as pro-oxidant, the susceptibility for LPO was flow-cytometrically assessed using the probe Bodipy581/591-C11. Sperm motility, monitored with a Qualisperm motility analyzer, increased after SLC treatment (P < .001). No significant correlations were found between motility and induced LPO with ferrous sulfate. The SP and extenders, per se, did not have a significant protective effect against LPO, but the interaction between SP and Kenney increased the susceptibility to LPO. However, the selected spermatozoa through Androcoll-E and the subsequent dilution in INRA had a significant protective effect against LPO (P < .05), especially when the oxidative insults were higher (80 μM). [Copyright &y& Elsevier]

• Alpaca ejaculates were liquified by gentle pipetting in Tris-citrate-fructose buffer. • Sperm were separated from seminal plasma with or without colloid centrifugation. • Motility and membrane integrity were greater after colloid centrifugation than in controls. • Sperm quality in colloid centrifuged samples was maintained during 24h cold storage. • Sperm quality in thawed samples was also greater after colloid centrifugation but the freezing protocol requires optimization. Viscous camelid ejaculates present problems for sperm handling and sperm preservation. In the present study, a technique that had been used for dromedary camel semen was tested with alpaca semen. Ejaculates (n=9) were collected by artificial vagina at San Marcos University, Lima, and were liquefied by gentle pipetting in tris-citrate-fructose. Half of the sample was prepared by Single Layer Centrifugation (SLC) through a colloid; the other half was centrifuged without colloid as a control. Each control and SLC sample was then split into two parts; one part was stored cooled for 24 h at 5 °C and the other part was frozen, resulting in 4 treatments for each ejaculate. All samples were evaluated for sperm motility, hypoosmotic swelling test (HOST), plasma membrane integrity, and morphology, immediately after centrifugation and again after storage Total motility and plasma membrane integrity were greater in samples prepared by SLC than controls (motility 72±13% vs. 57±7%; plasma membrane integrity 63±13% vs. 54±8%, for SLC and controls respectively). Normal morphology and HOST were not different between treatments (65±13 vs. 61±13% and 42±6 vs. 39±10%, for SLC and controls respectively). After 24 h cooled storage, motility and plasma membrane integrity were greater for SLC samples (motility: 51±16 vs. 34±15%; p<0.001; membrane integrity: 51±15 vs. 40±18%; P < 0.05 for SLC and controls, respectively); HOST (40±14 vs. 34±11%) and normal morphology (67±13 vs. 63±14%) were not different between treatments. Sperm quality decreased considerably after cryopreservation (P <0.001 for all parameters); however, motility (P <0.01), plasma membrane integrity (P <0.05) and morphology (P <0.05) were higher for SLC than for controls. These results indicate that alpaca spermatozoa can be extracted from semen using a combination of pipetting and SLC, potentially with a beneficial effect on sperm quality. Samples could be stored cooled for 24 h, retaining better motility than controls; motility and plasma membrane integrity were greater in SLC samples than controls after freezing and thawing but the freezing protocol requires improvement. [ABSTRACT FROM AUTHOR]

Removal of seminal plasma facilitates stallion sperm survival during storage, but washing may damage sperm chromatin. Therefore, sperm quality was compared in samples following single-layer centrifugation (SLC) or sperm washing and controls (extension only) in two extenders, INRA82 and INRA96. Ejaculates from six stallions were split among six treatments: SLC, sperm washing, and controls, in INRA82 and INRA96. Sperm motility and acrosome status were evaluated at 0, 24, 48, 72 and 96 hours; morphology at 0, 24, 48, 72 hours and chromatin integrity at 0 and 96 hours, with storage at 6°C. Sperm samples in INRA96 had better motility, acrosome status, and normal morphology than samples in INRA82. The SLC samples had higher motility and fewer reacted acrosomes than controls, and lower fragmented chromatin than washed samples. Fewer spermatozoa with tail defects were observed after SLC than after sperm washing; spermatozoa washed in INRA82 had fewer tail defects than those washed in INRA96. In conclusion, sperm quality (except for morphology) was better in INRA96 than in INRA82 and was better in SLC samples than in washed samples or controls. The SLC method is a useful adjunct to stallion sperm preparation, especially for storage before artificial insemination. • Washing, colloid centrifugation and simple extension of stallion semen were compared. • Sperm quality was better after colloid centrifugation than sperm washing or extension. • Sperm quality was better in INRA96 than INRA82 except for sperm morphology. • Colloid centrifugation could be a useful technique when preparing semen for artificial inseminations (AI). [ABSTRACT FROM AUTHOR]

Stallion sperm was vitrified using straws in comparison with spheres and conventional freezing. Vitrification was performed plunging 30 μL of sperm (spheres) or 0.5 mL straws into liquid nitrogen (LN 2) and conventional freezing using 0.5 mL straws frozen in LN 2 vapors. Sperm vitrified in straws were submitted to different warming procedures (42°C/20 seconds; 60°C/15 seconds) and single-layer centrifugation (SLC). Total (TM, %) and progressive sperm motility (PM, %), plasma membrane (IMS, %) and acrosome integrity (AIS, %) were statistically compared between treatments (mean ± SEM). Significant higher values (P <.001) were obtained after vitrification using spheres in comparison with conventional freezing and vitrification in straws for TM (54.46 ± 3.2 vs. 36.47 ± 3.2 vs. 2.50 ± 1.2, %), PM (38.63 ± 3.4 vs. 15.11 ± 2.0 vs. 1.9 ± 0.9, %), IMS (65.40 ± 2.8 vs. 50.50 ± 2.8 vs. 21.63 ± 2.1, %), and AIS (48.89 ± 2.8 vs. 15.46 ± 1.7 vs. 4.69 ± 0.9, %). No differences were found between warming procedures. Single-layer centrifugation after warming at 42°C/20 seconds obtained higher values (P <.05) than unselected samples for TM (32.52 ± 5.8%), PM (14.22 ± 2.8%), IMS (60.01 ± 3.2%), and AIS (44.5 ± 2.2%), whereas selection after 60°C/15 seconds increased TM (23.11 ± 4.3%) and IMS (67.11 ± 3.9%). In conclusion, vitrification in spheres obtained better sperm quality than conventional freezing and vitrification in straws. Warming procedures did not affect the sperm quality but SLC could be a strategy to enhance the quality of the samples after sperm vitrification using 0.5 mL straws. • Vitrification in spheres showed better sperm quality than conventional freezing. • Vitrification using 0.5 mL straws resulted in poor sperm quality after warming. • Warming procedures of vitrified straws (42°C/20 seconds; 60°C/15 seconds) showed no differences in sperm quality. • Single-layer centrifugation improved the sperm quality after vitrification and warming. [ABSTRACT FROM AUTHOR]

The presence of bacteria poses a significant challenge to the quality of stallion semen used in artificial insemination. The bacterial content of insemination doses arises from various sources, such as the healthy stallion, environment, and collection equipment, and is implicated in fertility problems as well as reduced sperm quality during storage. The conventional approach of adding antibiotics to semen extenders raises concerns about antimicrobial resistance and potential negative effects on sperm characteristics, and may not be effective in inhibiting all bacteria. The objective of this study was to determine whether an innovative alternative to antibiotic usage – centrifugation through a single layer of a low density colloid (SLC) – could reduce the bacterial load in stallion semen, and to compare sperm characteristics in samples arising from this procedure, or simple extension of the ejaculate in semen extender, or from sperm washing, i.e. adding extender and then centrifuging the sample to allow the removal of most of the seminal plasma and extender. Eighteen semen samples were collected from six stallions. The semen samples were split and extended prior to washing or SLC, or received no further treatment other than extension. After preparation aliquots from each type of sample were sent for bacteriological examination; the remaining samples were stored for up to 72 h, with daily checks on sperm quality. The low density colloid SLC outperformed sperm washing or extension for bacterial reduction, effectively removing several bacterial species. The bacterial load in the samples was as follows: extended semen, 16 ± 6.7 × 105; washed, 5.8 ± 2.0 × 105; SLC, 2.3 ± 0.88 × 105, p < 0.0001. In addition, SLC completely removed some bacterial species, such as Staphylococcus xylosus. Although there is no selection for robust spermatozoa with the low density colloid, sperm motility, membrane integrity, and DNA fragmentation were not different to washed sperm samples. These findings suggest that SLC with a low density colloid offers a promising method for reducing bacterial contamination in stallion semen without resorting to antibiotics. • Stallion ejaculates were prepared without antibiotics by extension, washing or SLC. • Bacterial load was highest in extended semen and lowest in SLC samples. • Sperm characteristics were not different between the three treatments. • Total motility was less in extended samples than in other treatments at 72 h. • SLC through a low density colloid could be an alternative to antibiotics. [ABSTRACT FROM AUTHOR]

The storage of boar semen samples at 17 °C for artificial insemination (AI) doses enables the proliferation of the bacteria, making antibiotics necessary. This can contribute to the development of antimicrobial resistance (AMR). This study tested bacterial presence and sperm chromatin structure after using a low-density colloid (Porcicoll) as an antibiotic alternative to eliminate bacteria. Ejaculates (8 boars, 3 ejaculates each) were split as control and low-density colloid centrifugation (single layer centrifugation, SLC, 20%, and 30% Porcicoll) into 500 ml tubes. Analyses were carried out at days 0, 3, and 7 (17 °C) for microbial presence and sperm chromatin structure analysis: %DFI (DNA fragmentation) and %HDS (chromatin immaturity), monobromobimane (mBBr; free thiols and disulfide bridges), and chromomycin A3 (CMA3; chromatin compaction). Besides comparing bacterial presence (7 species identified) and chromatin variables between treatments, the associations between these sets of variables were described by canonical correlation analysis (CCA). Results showed a significant decrease of some bacteria or a complete removal after SLC (especially for P30). SLC also caused a decrease of %HDS and an increase of disulfide bridges and low and medium mBBr populations, suggesting the removal of immature sperm (poor chromatin compaction). CCA showed an association pattern compatible with the degradation of sperm chromatin parameters with bacterial contamination, especially Enterobacteria, P. aeuriginosa, and K. variicola. In conclusion, bacterial contamination affects sperm chromatin beyond DNA fragmentation; SLC with low-density colloid not only removes bacteria from boar semen, but also chromatin structure is enhanced after selection. • Antibiotics in semen extenders might contribute to antimicrobial resistance. • Bacteria could be removed from boar spermatozoa by low-density Porcicoll colloid. • Porcicoll improved the overall sperm chromatin structure by removing. • Some benefits of Porcicoll on sperm chromatin could be due to a direct effect. • Canonical correlation analysis (CCA) helped the multiparametric interpretation. [ABSTRACT FROM AUTHOR]

In endangered animals that have been found dead or sterilized for medical reasons, testis is the ultimate source of haploid DNA or sperm. Thus, preservation of testicular sperm may be performed to rescue their genetics. The aim of this study was to evaluate protocols for testicular sperm freezing: as tissue fragments or cell suspension in domestic cats as a model. A pair of testes from each cat (n = 9) were cut into eight equal pieces. Four randomly selected pieces were cryopreserved as: (1) tissue pieces using two-step freezing; (2) tissue pieces using a slow passive cooling device (CoolCell); (3) sperm suspension after single-layer centrifugation (SLC) through colloids; and (4) sperm suspension without being processed through SLC. A testicular piece from each cat served as fresh control. Testicular sperm membrane and DNA integrity were evaluated before, and after, the cryopreservation process. In addition, spermatogenic cell types (testicular sperm, spermatogonia, spermatocyte, and spermatid) present in the suspension samples were counted before and after SLC. The results found that testicular sperm membrane integrity in the suspension after SLC process was higher than that in the fragment form neither using the two-step nor CoolCell freezing, both before and after freezing (before freezing: 92.3 ± 3.4 vs. 81 ± 4.5 and 80.0 ± 7.0; after freezing: 84.5 ± 4.6 vs. 71.2 ± 12 and 76.2 ± 4.6; P ≤ 0.05). Testicular sperm DNA integrity was, however, not different among groups. Furthermore, the samples processed through the SLC had higher ration of sperm cells: other spermatogenic cells than those were not processed through the SLC (88.9 ± 3.8 vs. 30 ± 7.9; P ≤ 0.05). In summary, testicular sperm cryopreserved as a minced suspension is considered suitable in terms of preventing sperm membrane integrity, and SLC is considered a selection tool for enriching haploid sperm cells from castrated or postmortem cats. [ABSTRACT FROM AUTHOR]

In the dairy breeding industry, prediction of bull fertility in artificial insemination (AI) is important for efficient and economically sustainable production. However, it is challenging to identify bulls with superior fertility applying conventional in vitro sperm assays. In the present study, sperm functionality was investigated to identify a multivariate model that could predict fertility. Two groups of young Norwegian Red bulls were selected, one with inferior fertility (18 bulls) and one with superior fertility (19 bulls) based on non-return rate after 56 days (NR56). Frozen-thawed semen doses were analysed for sperm chromatin integrity, viability, acrosome integrity, motility, and ATP content. A targeted approach was used to study intracellular concentrations of amino acids and trace elements in viable sperm cells. Significant differences between the two groups of bulls were observed, both for sperm functional attributes and intracellular concentrations of metabolites. Pearson correlation analyses indicated a negative relationship between NR56 and chromatin integrity parameters, DNA fragmentation index (DFI) and high DNA stainability (HDS). Several motility parameters correlated positively with NR56. The concentrations of cysteine and glutamic acid in sperm cells correlated negatively with NR56, while the concentrations of aspartic acid, leucine and serine showed a positive NR56-correlation. The sperm intracellular concentrations of the trace elements Fe, Al and Zn, correlated negatively with NR56. Correlations were observed between several sperm parameters and metabolites. Stepwise multiple regression analysis indicated that the best predictor of NR56 was a model containing %DFI, together with the intracellular sperm concentration of aspartic acid, Fe and Zn. This model explained 59% of the variability in NR56. • Bulls of contrasting NR56 differed in sperm quality parameters and metabolome. • Metabolite analyses was performed on viable frozen-thawed sperm cells. • A model including DFI, aspartic acid, Fe and Zn explained variation in bulls' NR56. [ABSTRACT FROM AUTHOR]

Abstract The objective of this study was to evaluate the effect of different freezing rates and thawing temperatures on the post-thaw quality of camel spermatozoa. Ten ejaculates from five male camels were frozen at five different freezing rates, achieved by placing the straws at specific heights above the surface of liquid nitrogen for different lengths of time (4 cm for 15 min; 1 cm for 15 min; 7 cm for 15 min; 7 cm for 5 min + 4 cm for 3 min; 4 cm for 5 min + 1 cm for 3 min) followed by storage in liquid nitrogen. Two thawing temperatures (37° for 30 s and 60 °C for 10 s) were subsequently tested. Post-thawing, the samples were evaluated for total and progressive motility, kinematics, membrane and acrosome integrity, and membrane functionality (hypoosmotic swelling test) at zero and 1 h post thawing. Total and progressive motility were significantly higher for the fastest freezing rate (at 1 cm) at 0 h (p < 0.01 for both), as were VCL (p < 0.01), VSL (p < 0.05) and STR (p < 0.05). Freezing at 4 cm produced the lowest values of STR compared to other treatments (p < 0.05). At 1 h, no differences in total motility were observed between freezing at 4 cm and 1 cm, both being significantly better than freezing rate 7 cm + 4 cm (p < 0.01). For progressive motility and VSL, only freezing at 1 cm was superior to the 7 cm + 4 cm combination (p < 0.001 and p < 0.05 respectively). Membrane integrity at 1 h was higher for freezing at 7 cm than at 1 cm (p < 0.01). For thawing temperatures, total motility and progressive motility at 0 h and 1 h (p < 0.001), and acrosome integrity at 1 h (p < 0.01) were higher for 60 °C thawing temperature than 37 °C. The kinematics VCL (p < 0.001), VSL and STR (p < 0.01), and VAP (p < 0.05) showed higher values for 60 °C thawing temperature than 37 °C at 0 h. After 1 h, higher values for VSL, VCL and VAP (p < 0.05) were observed for 60 °C than for 37 °C. In conclusion, a fast freezing rate would probably be beneficial for camel semen, and thawing should be conducted at 60 °C. [ABSTRACT FROM AUTHOR]

Conventionally inseminated spermatozoa suffer a dramatic reduction in numbers during their long journey until fertilization. In addition sperm survival seems to be strongly affected by the reconstitution of the female reproductive tract in the post partum period. The purpose of this study was to develop a novel AI technique for cattle that allows the deposition of spermatozoa directly into the ampulla in the immediate vicinity of the fertilization site. This new reproductive biotechnique was investigated with focus on semen origin, sperm dosage, semen preparation and time of insemination. Finally, a first practical application was carried out by inseminating superovulated heifers with sex-sorted semen. In total, 49 Simmental heifers were used for 65 intratubal inseminations (ITI) with single ovulation and 8 ITIs after superovulation, respectively. Insemination into the oviduct was performed under epidural anesthesia via transvaginal endoscopy using a curved glass capillary loaded with semen. Two days later the oviduct and the adjacent uterine horn were endoscopically flushed and embryos or unfertilized oocytes were collected for determination of fertilization success. Across all experimental groups, tubal insemination successfully resulted in the collection of embryos; however, first tubal AI attempts and ITIs close to ovulation led to low recovery rates. In total, 109 complexes were flushed from ITIs in superstimulated heifers (n = 8) using sex sorted semen, of which 24 (22%) were at the embryo stage. In conclusion, it was shown that intratubal insemination can be successfully used for semen deposition, thus bypassing the lower female genital tract. Factors such as time of insemination, semen processing and semen quantity for superovulatory use should be further investigated. [ABSTRACT FROM AUTHOR]

Seminal plasma (SP) contains proteins that may influence cryosurvival and prevent capacitation-like changes due to freezing and thawing. The objective of this study was to investigate the effect of adding pooled SP from “good” (GF) or “bad” (BF) freezer stallions on sperm cells' fertilizing ability. “Good freezers” refers to stallions that usually produce ejaculates which can withstand cryopreservation, whilst “bad freezer” stallions produce ejaculates which cannot tolerate the freezing process. A heterologous zona binding assay with in vitro matured bovine oocytes was used to assess the binding ability of equine sperm cells as a possible alternative to artificial insemination trials. The effect of adding SP i) prior to cryopreservation; ii) after thawing of sperm cells selected by single layer centrifugation (SLC); iii) to capacitation medium, was evaluated. Adding SP from GF stallions prior to cryopreservation reduced the mean number of sperm cells bound to the zona pellucida (ZP) compared to control (P = 0.0003), SP-free sperm cells and group received SP from BF stallions (P ≤ 0.0001 for both). After thawing SLC-selected sperm cells treated with 5% SP showed a decrease in binding ability compared with SP-free sperm cells (P ≤ 0.0001). The binding affinity of sperm cells was higher in the group treated with SP from GF than with SP from BF stallions (P ≤ 0.05). Prolonged exposure to SP impaired the ability of stallion sperm cells to undergo capacitation and bind to ZP, regardless of the source of SP (P ≤ 0.0001). The response of equine sperm cells to SP is influenced by the ability of the sperm cells to withstand cryopreservation and is affected by the timing of exposure and the origin of SP. Customization of the protocol for individual stallions is recommended to optimize the effect. [ABSTRACT FROM AUTHOR]

Sperm samples may be used for assisted reproductive technologies (e.g., farmed or endangered species) or as a source of haploid DNA or sperm-specific RNA. When ejaculated spermatozoa are not available or are very difficult to obtain, as is the case for most wild endangered species, the epididymides of dead animals (e.g., animals that have been found dead, shot by hunters or poachers, or that that require euthanasia in zoological collections) can be used as a source of sperm. Such epididymal sperm samples are usually contaminated with cellular debris, erythrocytes, leukocytes, and sometimes also bacteria. These contaminants may be sources of reactive oxygen species that damage spermatozoa during freezing or contribute undesired genetic material from diploid cells. We used single-layer centrifugation through a colloid formulation, Androcoll-C, to successfully separate wolf epididymal spermatozoa from contaminating cells and cellular debris in epididymal samples harvested from carcasses. Such a procedure may potentially be applied to epididymal sperm samples from other species. [ABSTRACT FROM AUTHOR]

• Thawed boar semen samples were split between MACS and SLC treatment groups. • Membrane integrity and mitochondrial potential were higher in SLC samples than controls. • MACS selected samples had fewer spermatozoa with immature chromatin than controls. • In general, sperm quality was better in SLC samples than in MACS selected samples. Sperm selection techniques, such as magnetic activated cell sorting (MACS) and colloid centrifugation, are reported to select good quality spermatozoa from semen samples of various species. Although the sperm quality of fresh boar semen is usually good, cryopreservation has a negative effect on parameters such as plasma membrane integrity and mitochondrial activity. Therefore, the objective of the present study was to determine whether MACS or centrifugation through a single layer of colloid (Single Layer Centrifugation, SLC) would be beneficial in enriching thawed boar sperm samples for viable spermatozoa with active mitochondria and good chromatin integrity. Frozen samples from three boars, three ejaculates per boar, were thawed and split. One part was selected by MACS, one was prepared by SLC, and the remainder served as the control. Controls and the selected sperm samples were evaluated for sperm quality (plasma membrane integrity, chromatin integrity, mitochondrial membrane potential and production of reactive oxygen species). Although several aspects of sperm quality were improved in the SLC-selected sperm samples compared to control, the flow-through MACS samples were only improved in having a lower proportion of spermatozoa with immature chromatin (Hi green fluorescence) compared to the labeled control. Sperm quality in the SLC samples was better than in the flow-through samples from MACS. Therefore, despite promising reports of the use of MACS for selecting good quality spermatozoa from semen in other species, the method was not useful for improving sperm quality in the thawed boar sperm samples in this experiment. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: The virus porcine circovirus type 2 (PCV2) is associated with different disease entities, including reproductive failure. The objective of this study was to investigate the use of a semen processing technique for the elimination of infectious PCV2 in semen. PCV2 was chosen as a model virus because of its small size, high resistance to inactivation and as a known risk factor for boar semen contamination. Aliquots of ejaculates were spiked with PCV2 and processed by a double processing technique, consisting of Single Layer Centrifugation on Androcoll™-P followed by a “swim-up” procedure. Samples were collected from the resulting fractions during the selection process and analyzed for the presence of infectious PCV2. Virus titres were determined by performing a 50% tissue culture infective dose assay (TCID50) by end point dilution and with the use of an indirect peroxidise monolayer assay technique. With an initial infectious virus titre of 3.25–3.82 (TCID50)/50μL the two-step sperm selection method eliminated 2.92±0.23 logs of infectious PCV2, corresponding to more than 99% reduction. Sperm quality was not affected by the selection procedure. [Copyright &y& Elsevier]

• 10-month old bulls can serve as semen donors but sperm quality may be poor or cryosurvival low. • Seminal plasma was collected from 10-month old bulls and again later. • Fertility-associated proteins were evaluated by fast protein liquid chromatography. • Heparin-binding proteins were more abundant in Sample II than in Sample I. Breeding companies want to use semen from bulls as soon as possible to take advantage of their desirable genetics. It takes several weeks for the sperm quality of young bulls to stabilize and for post-thaw sperm quality to become acceptable for artificial insemination. Seminal plasma proteins protect spermatozoa during cryopreservation; it may take some time for the seminal plasma protein profile to stabilize. The purpose of this study was to determine if the seminal plasma protein profile can be used as a marker of likely seminal maturity in young bulls. A comparison was made of the seminal plasma protein profile in the ejaculates of 10 bulls of 9-10 months old (Sample I), with the profiles from ejaculates taken from the same bulls at 13-16 months old (Sample II) using fast protein liquid chromatography. This is a method for separating classes of proteins according to their binding ability. The peak area and peak height of different classes of proteins did not differ significantly between the two samples for each bull, except for peak 5 (heparin-binding proteins) and total peak area (p<0.05). The heparin-binding protein peak height and area were significantly higher (p<0.05) in Sample II than in Sample I. In conclusion, levels of fertility associated heparin-binding proteins increase with age in young bulls and might serve as a biomarker of sperm maturity.. [ABSTRACT FROM AUTHOR]

• Seminal plasma (SP) regulates immune responses in the reproductive tract via cytokines. • Cytokine production by endometrial cells may thus depend on bull fertility. • Endometrial cells in culture were challenged with SP from high and low fertility bulls. • TGF-β1, TGF-β2, TGF-β3, IL-6 and IL-8 were measured after challenge with SP. • More cytokine production was stimulated by SP from low fertility bulls than high. Seminal plasma (SP) regulates immune responses in the female reproductive tract through specific cytokines. It is not known whether SP from high fertility bulls (H) differs from SP from low fertility bulls (L). In this study, the cytokine response of bovine endometrial epithelial cells (bEEC) in culture was investigated after challenge with SP from two bulls of below average (L) or three bulls of above average fertility (H). The bEECs were challenged with 1% or 4% SP from l - or H-fertility bulls (L1, L4, H1, H4, respectively) or 1% or 4% PBS as control (C1, C4) for 72 h. The culture media were analysed for concentrations (pg/million cells) of transforming growth factor beta (TGF-β1, TGF-β2 and TGF-β3) by Luminex, and Interleukin 6 and 8 (IL-6, IL-8) by ELISA. Challenge significantly affected production of TGF-ß1, TGF-ß2 and IL-8 compared to controls and was affected by bull fertility (p < 0.0001), SP concentration (p < 0.0001) and their interaction (p < 0.0001). A higher production of TGF-β1, TGF-β2 and IL-8 (p < 0.0001), and also IL-6 (p < 0.01), resulted from challenge with high doses of SP, being higher for L than H (p < 0.05). For TGF-β3, fertility of bull (p < 0.05). For TGF-B3, fertility of bull (p < 0.05) and the interaction between fertility and concentration of SP were significant (p < 0.01). In conclusion, 4% SP from L bulls stimulated more TGF-β1, TGF-β2, TGF-β3, IL-6 and IL-8 production than SP from H bulls, indicating that stimulation of the endometrium is relevant for fertility. Seminal plasma from high fertility bulls seems to affect cytokine production in utero positively in inseminated cows. [ABSTRACT FROM AUTHOR]