Sperm quality variables of sex-sorted bull semen produced by magnetic-activated cell sorting coupled with recombinant antibodies targeting Y-chromosome-bearing sperm.

The immunological sexing method using antibodies offers cost-effective, high-volume production but faces challenges in terms of X-sperm purity in sexed semen. This research aimed to produce sexed bull semen using highly specific recombinant antibodies in magnetic-activated cell sorting (MACS), evaluate sperm quality and kinematic parameters, and verify the sex ratio of sperm, embryos, and live calves. Fresh semen from two Angus bulls was separated into two equal groups: conventional (CONV) semen and semen sexed using MACS with Y-scFv antibody conjugation to separate two fractions, i.e., the X-enriched and Y-enriched fractions. Then, computer assisted semen analysis and imaging flow cytometry were used to evaluate sperm motility and kinematic variables, acrosomal integrity, sperm viability, and sperm sex ratios. The results showed that sperm motility and quality did not differ between X-enriched and CONV semen. However, the Y-enriched fraction showed significantly lower sperm quality than the X-enriched fraction and CONV semen. The sperm ratio revealed that X-sperm accounted for up to 79.50% of the X-enriched fraction, while Y-sperm accounted for up to 78.56% of the Y-enriched fraction. The sex ratio of embryos was examined using in vitro fertilization. The cleavage rates using CONV and X-enriched semen were significantly higher than that using Y-enriched semen. Accordingly, 88.26% female blastocysts were obtained by using X-enriched semen, and 83.58% male blastocysts were obtained by using Y-enriched semen. In farm trials, 304 cows were subjected to AI using X-enriched and CONV semen. The pregnancy rate did not differ between the X-enriched and CONV semen groups. On the other hand, X-enriched semen generated significantly more live female calves (83.64%) than CONV semen (47.00%). The MACS sexing method significantly enhanced the X-sperm purity in sexed semen, producing high-quality sperm, a high percentage of female blastocytes, and a high percentage of live female calves.

Evaluation of sexed semen-based artificial insemination in Tharparkar cattle under organized farm condition.

Sexed semen facilitates additional female calf production for the expansion of a herd at a faster rate and also curtails the surplus production of unwanted male calves. A study was conducted to evaluate the performance of sexed semen in indigenous Tharparkar cows based on 114 artificial inseminations (AI) performed at natural oestrus using two protocols i.e., single AI (n = 48) and double AI (n = 66). Overall, the first service conception rate (CR) was significantly higher in double (53.0%) than single (33.3%) AI protocol. The odds ratio of conception rate in double AI was 2.26 (χ2 = 4.4, df = 1, p = .04) with respect to single AI. The time that elapsed since the detection of oestrus to insemination was also analysed. In a single AI protocol, the CR was higher (p < .05) at 16 h (54.6%) than insemination at 8 h (27.0%) following the onset of oestrus. Yet, the CR using double AI protocol did not differ (p = .73) significantly when AIs were performed either at 8 h and 24 h (51.9%) or 16 h and 24 h (57.1%) post onset of oestrus. Besides, like the single AI protocol, the parity of the animals also influenced the CR, being higher in heifers (n = 22) than those of parous (n = 92) cows (72.73 vs. 40.43%, χ2 = 7.48, df = 1, p = .006) in the present study. The odds ratio of conception in heifers was 3.93 with respect to parous cows. Overall, the birth of female calf was 91.7%. In conclusion, the present study indicates a future promise of the sexed semen for the production of more female offspring from Tharparkar cattle.

Biochemical Features of X or Y Chromosome-Bearing Spermatozoa for Sperm Sexing.

This review presents information on biochemical features of spermatozoa bearing X or Y chromosome, enabling production of a sperm fraction with pre-defined sex chromosome. The almost only technology currently used for such separation (called sexing) is based on the fluorescence-activated cell sorting of sperm depending on DNA content. In addition to the applied aspects, this technology made it possible to analyze properties of the isolated populations of spermatozoa bearing X or Y chromosome. In recent years, existence of the differences between these populations at the transcriptome and proteome level have been reported in a number of studies. It is noteworthy that these differences are primarily related to the energy metabolism and flagellar structural proteins. New methods of sperm enrichment with X or Y chromosome cells are based on the differences in motility between the spermatozoa with different sex chromosomes. Sperm sexing is a part of the widespread protocol of artificial insemination of cows with cryopreserved semen, it allows to increase proportion of the offspring with the required sex. In addition, advances in the separation of X and Y spermatozoa may allow this approach to be applied in clinical practice to avoid sex-linked diseases.

Characterization of semen type prevalence and allocation in Holstein and Jersey females in the United States.

Our objective was to characterize semen type prevalence and allocation to inseminate US Holstein and Jersey females by year, parity, service number, and herd size. A secondary objective was to identify the prevalence of beef breed sires selected to create beef × Holstein and beef × Jersey crossbred calves. The final data set included 8,244,653 total inseminations of 4,880,752 Holstein females across 9,155 herds, and 435,267 total inseminations of 266,058 Jersey females across 2,759 herds from October 2019 to July 2021. This data set represents approximately 42 and 27% of the total dairy cows and heifers, respectively, across approximately 40% of the total licensed dairy herds in the continental United States. Holstein and Jersey females were inseminated with 1 of 4 semen types: (1) beef, (2) conventional, (3) sexed, or (4) other dairy. The top 4 beef breeds used to produce beef × Holstein and beef × Jersey crossbred calves, respectively, were Angus (55.1 and 39.1%), Limousin (13.9, and 23.5%), Simmental (11.7 and 20.5%), and Crossbreed Beef (11.3 and 4.8%). From 2019 to 2021, the use of sexed semen to inseminate Holstein and Jersey females increased from 11.0 and 24.5% to 17.7 and 32.1%, respectively, and the use of beef semen to inseminate Holstein and Jersey females increased from 18.2 and 11.4% to 26.1 and 21.2%, respectively. The use of beef semen to inseminate Holstein and Jersey females increased with increasing parity and service number, whereas the use of sexed semen decreased with increasing parity and service number supporting that farmers used sexed semen more aggressively in higher fertility and younger females with greater genetic merit. Overall, the increase in sexed and beef semen inseminations was driven primarily by larger herds. In conclusion, sexed and beef semen inseminations in US Holstein and Jersey females increased from 2019 to 2021 and was allocated differentially based on parity and service number. This increase was driven primarily by larger dairy herds possibly due to differences in reproductive performance and economies of scale.

Relationship between the timing of insemination based on estrus detected by the automatic activity monitoring system and conception rates using sex-sorted semen in Holstein dairy cattle.

We investigated the optimal timing of artificial insemination (AI) for achieving pregnancy according to the onset/end of estrus detected by an accelerometer system in Holstein cattle. The conception rates of conventional semen were used as a reference. The conception rate from AI of sex-sorted semen was higher at -4 to 4 h (57.1%) from the end of estrus than those at -12 to -4 h (37.7%) and 12-20 h (30.3%), whereas AI at 4-12 h showed an intermediate conception rate (47.4%). Conversely, conception rates were similar in AI performed between 0 and 32 h from the onset of estrus. Regarding conventional semen, the interval from the onset and end of estrus did not affect conception rates. The present results suggest that the time of the end of estrus is the better indicator of optimal AI timing for sex-sorted semen than the onset of estrus.

Economics of timed artificial insemination with unsorted or sexed semen in a high-producing, pasture-based dairy production system.

This study used a stochastic simulation model to estimate the potential economic benefit of using timed artificial insemination (TAI) in combination with conventional unsorted (TCONV) and sexed (TSEX) semen in heifers only (TCONV-H, TSEX-H) and in both heifers and lactating cows (TCONV-HC, TSEX-HC) in a high-producing, pasture-based production system. The scenarios were compared with a conventional reproductive policy (CONV) in which heifers and cows were inseminated with conventional unsorted semen after estrus detection. Sensitivity analysis was also used to estimate the effect of hormone costs from TAI use on the profitability of each program relative to CONV. The mean annual (± standard deviation) profit advantage (ΔPROF) over CONV for TCONV-H, TCONV-HC, TSEX-H, and TSEX-HC scenarios were €3.90/cow ± 4.65, €34.11/cow ± 25.69, €13.96/cow ± 6.83, and €41.52/cow ± 42.86, respectively. Combined application of both technologies was shown to return a greater annual ΔPROF on average compared with that achievable from TAI alone. However, the risk of not returning a positive annual ΔPROF varied across the scenarios with higher risk in TCONV-H and TSEX-HC. Specifically, TCONV-H and TSEX-HC had a 24 and 18% chance, respectively, of not returning a positive annual ΔPROF. Sensitivity analysis showed that when hormone costs increased by €10/cow TCONV-H and TSEX-HC had a 38 and 23% chance, respectively, of not returning a positive annual ΔPROF. The range in ΔPROF for TCONV policies was most sensitive to the TAI pregnancy rate and TSEX policies were most sensitive to the relative fertility achieved with sexed compared with unsorted semen. This study has shown TAI and sexed semen are complementary technologies that can increase genetic gain and profitability in a pasture-based, dairy production system.

Applications and world-wide use of sexed semen in cattle.

Successful sorting of sperm based on presence of the X- or Y-chromosome was first reported in the early 1980's with the first live births reported in rabbits in 1988. Subsequent development of technological efficiencies resulted in commercialization of sex-sorted semen to cattle producers in 2003-2005. At product launch, low throughput dictated that reasonable prices to the producer could only be accomplished with extremely low sperm number dosages (2 × 106). Furthermore, conception rates were 70%-75% of those achieved by conventional unsorted product. Refinements in sorting equipment have enhanced the number of sperm that can be sorted from a semen sample and (or) aliquot of time, which translates into reduced production costs, while modifications to other aspects of sperm processing and freezing have facilitated maintenance of a conception potential more similar to that of conventional semen. More recently, strategic use of sex-sorted semen coupled with genomic technologies to identify superior females to satisfy replacement female needs has, by default, led to identification of a population of dairy cows from which replacements are not desired, leading to a tremendous increase in use of beef semen in dairy herds. Though exact numbers are unavailable, estimates indicate sex-sorted semen is rapidly approaching 30% of the total AI market share in North America. Though the primary application of sex-sorted semen is to accelerate genetic progress while enhancing biosecurity through in-house production of replacement animals, numerous other potential applications are evolving or are under consideration.

Dairy cattle farmers' preferences for different breeding tools.

Breeding technologies play a significant role in improving dairy cattle production. Scientifically proven tools for improved management and genetic gain in dairy herds, such as sexed semen, beef semen, genomic testing, dairy crossbreeding, and multiple ovulation embryo transfer (MOET), are readily available to dairy farmers. However, despite good accessibility, decreasing costs, and continuous development of these tools, their use in Sweden is limited. This study investigated Swedish dairy farmers' preferences for breeding tools through a survey including a discrete choice experiment. The survey was distributed online to 1 521 Swedish farmers and by an open link published through a farming magazine. In total, the study included 204 completed responses. The discrete choice experiment consisted of 10 questions with two alternative combinations, which gave 48 combinations in total. Utility values and part-worth values were computed using a conditional logit model based on the responses in the discrete choice experiment for nine groups of respondents: one group with all respondents, two groups based on respondents using dairy crossbreeding or not within the past 12 months, two based on herd size, two based on respondent age, and two based on whether respondents had used breeding advisory services or not. The strongest preferences in all groups were for using sexed semen and beef semen. Genomic testing was also significantly preferred by all groups of respondents. Except in large herds, MOET on own animals was significantly and relatively strongly disfavoured by all groups. Buying embryos had no significant utility value to any group. Dairy crossbreeding had low and insignificant utility values in the group of all respondents, but it was strongly favoured by the group that had used dairy crossbreeding within the past 12 months, and it was disfavoured by the group that had not. Part-worth values of combined breeding tools showed that combinations of sexed and beef semen, alone or with genomic testing without dairy crossbreeding, were the most preferred tools. Compared with the most common combinations of breeding tools used in the past 12 months, the part-worth values indicated that Swedish dairy farmers may prefer to use breeding tools more than they do today. Statements on the different breeding tools indicated that the respondents agreed with the benefits attributed to the breeding tools, but these benefits may not be worth the cost of genomic testing and the time consumption of MOET. These valuable insights can be used for further development of breeding tools.

Short-term storage of semen samples in acidic extender increases the proportion of females in pigs.

BACKGROUND: Sex preselection is a desired goal of the animal industry to improve production efficiency, depending on industry demand. In the porcine industry, there is a general preference for pork from female and surgically castrated male pigs. Therefore, the birth of more females than males in a litter leads to economic benefits and improved animal welfare in the pig production industry. Our previous study suggested that the porcine semen extender (BTS) adjusted to pH 6.2 maximises the differences in viability between X-chromosome-bearing (X) spermatozoa and Y-chromosome-bearing (Y) spermatozoa without affecting sperm's functional parameters. In this study we aimed to evaluate whether the pH 6.2 extender is applicable at the farm level for increasing the number of female piglets without a decline in spermatozoa fertility. Artificial insemination (AI) was carried out with spermatozoa stored at pH 6.2 and pH 7.2 (original BTS) at day 1 and day 2 of storage. Next, the functional parameters of the spermatozoa, litter size, farrowing rate, and female-to-male ratio of offspring were determined. RESULTS: Although sperm motility decreased significantly after 2 d of storage, the viability of spermatozoa was preserved at pH 6.2 for 3 d. There was no significant difference in the farrowing rate and average litter size between the group inseminated with the spermatozoa stored in (pH 7.2) and that inseminated with spermatozoa stored in acidic BTS. The percentage of female piglets was approximately 1.5-fold higher in sows inseminated on day 1 in the pH 6.2 than in the pH 7.2 group. Furthermore, although there was no significant difference in the female-to-male ratio, the percentage of female piglets born was slightly higher in the pH 6.2 group than in the pH 7.2 group on day 2. CONCLUSIONS: The method optimised in our study is simple, economical, and may enhance the number of female births without any decline in spermatozoa fertility.

Economic impact of different strategies to use sex-sorted sperm for reproductive management in seasonal-calving, pasture-based dairy herds.

To maximize efficiency, profitability, and societal acceptance of modern dairy production, it is important to minimize the production of male dairy calves with poor beef merit. One solution involves using sex-sorted sperm (SS) to generate dairy replacements and breeding all other cows to an easy-calving, short-gestation bull with good beef merit. We used the Pasture Based Herd Dynamic Milk Model to investigate the effect of herd fertility and use of SS on farm net profit in a herd of 100 cows. This was completed by simulating herds with differing fertility performance (good, average, poor), and differing farm reproductive management [conventional semen (CONV) or SS with varying pregnancy per artificial insemination (P/AI) relative to CONV (i.e., relative P/AI 100%, 85%, and 70%)]. As an additional consideration, the method of allocating SS to cows was also examined. The first option used SS on random heifers and cows (S). The second option used SS on heifers and targeted high-fertility cows (SSel). The final option was similar to SSel, but used a fixed-time artificial insemination (AI) protocol to facilitate AI on the farm mating start date (SSync). For CONV, dairy breed semen was used for AI until 50 animals were pregnant (50% chance of a female calf), whereas for S, SSel, or SSync the target number of animals successfully conceiving with SS was set at 28 (based on assumed 90% chance of a female calf from pregnancies derived from SS). Beef breed semen was used on all other dams. The results indicated that the biggest effect on farm net profit was not based on whether or not SS was used, but instead was most affected by the overall fertility performance of the herd. Total farm profit decreased by 10% between the good and average fertility herds, and decreased by a further 12% between the average and poor fertility herds. In almost all situations, when the relative P/AI with SS was ≥85%, use of SS led to an overall increase of the farm net profit. There was an economic benefit of using either SSel or SSync compared with S for the average and poor fertility herds but not for the good fertility herd, highlighting an interaction between SS P/AI and overall herd fertility as well as management practices. If the relative P/AI with SS was <70%, the use of SS led to a decrease in profitability in all simulations except for SSync, highlighting the importance of a good management strategy for use of SS. The findings in this study indicated that SS has significant potential to help facilitate greater integration between the dairy and beef production sectors, as well as increase farm profitability when used appropriately.

Pregnancy and offspring sex ratio following insemination with SexedULTRA and conventional semen in cows in a commercial beef operation.

Pregnancy rate per AI (PR/AI) and breeding season pregnancy rates between insemination with sexed semen (SS; at 18 hr after the onset of oestrus) and conventional semen (CS; at 12 hr after the onset of oestrus,) and offspring gender ratio between two groups were compared. Angus cross cows (n = 686, during 2019 and 2020 breeding seasons) were oestrus-synchronized using Select-Synch + CIDR protocol and were observed thrice daily for oestrus until 72 hr after PGF2α administration. Cows expressed oestrus (n = 513) were inseminated with either SS (n = 246; SexedULTRA 4M™; y chromosome-bearing sperm) or CS (n = 267). Cows (n = 173) that failed to express oestrus at 72 hr after PGF2α received 100 µg of GnRH and CS insemination concomitantly. Two weeks later, cows were penned with natural service sires (bull:cow ratio 1:25) for 45 days. Pregnancy was diagnosed 30 days after bull removal. Calves' gender was determined at birth. For cows that expressed oestrus, PR/AI did not differ (p > .1) between SS (65.0%) and CS (66.7%) groups. The overall PR/AI differed (p < .05) between SS (65.0%) and CS (56.4%) groups. The natural service PR differed (p < .001) but breeding season PR (p > .05) did not differ between SS vs. CS groups. Bull:heifer gender ratio following AI was 88:12 and 52:48 for SS and CS groups, respectively, with an overall 66:34 ratio. Bull:heifer gender ratio for the two breeding seasons was 79:21 and 52:48 for SS and CS groups, respectively, with an overall 62:38 ratio. In conclusion, the fertility of SS insemination at 18 hr after onset of oestrus was 97% of CS insemination at 12 hr after onset of oestrus. Though breeding season pregnancy did not differ between SS and groups, preferred calf gender was 25 percentage points greater for SS over CS application. The gender accuracy was 88%.

Comparison of pregnancy per AI of heifers inseminated with sex-sorted or conventional semen after oestrus detection or timed artificial insemination.

The objective of the study was to compare the fertility after using sex-sorted or conventional semen either with oestrus detection (EST) or timed artificial insemination (TAI) in Holstein heifers. Holstein heifers were randomly assigned to one of the following treatments in a 2 × 2 factorial design. Heifers in the EST group were inseminated with sex-sorted (n = 114) or conventional semen (n = 100) after spontaneous or induced oestrus. Heifers in the TAI, subjected to the 5-day Cosynch+Progesterone protocol (GnRH+P4 insertion-5d-PGF2α +P4 removal-1d-PGF2α -2d-GnRH+TAI), were inseminated with sex-sorted (n = 113) or conventional semen (n = 88). Statistical analyses were performed using PROC GLIMMIX procedure of SAS 9.4 (SAS Institute Inc., Cary, NC). Overall P/AI was 60.7% for EST and 54.2% for TAI regardless of types of semen and 68.1% for conventional and 48.9% for sex-sorted semen regardless of insemination strategies. Fertility of heifers inseminated with either sex-sorted (53.5%; 44.2%) or conventional (69.0%; 67.0%) semen did not differ between EST and TAI respectively. Besides, the interaction between the semen type and the insemination strategy was not significant for P/AI. The embryonic loss was significantly greater with sex-sorted semen (17.1%) compared to conventional semen (1.6%). There was no sire effect with sex-sorted semen on P/AI (52.6% vs. 46.2%) and embryonic loss (16.4% vs. 18.0%). As expected, sex-sorted semen resulted in more female calves (89.8% vs. 51.6%) than conventional semen. Thus, sex-sorted semen can be used with 5-day Cosynch+Progesterone protocol to eliminate the inadequate oestrus detection and to increase female calves born in dairy heifers.

Bovine semen sexing: Sperm membrane proteomics as candidates for immunological selection of X- and Y-chromosome-bearing sperm.

The use of sexed semen in dairy and beef farms ensures the production of animals of the desired sex, resulting in a reduction of costs and an improvement of environmental sustainability. Several methods have been developed over the years, but most of them were abandoned due to their limited efficacy. Currently, the only commercially available method for the separation of X- and Y-chromosome-bearing sperm is fluorescence-activated cell sorting. However, this technique is expensive and has limited usefulness for the industry, considering that it cannot produce doses of sexed semen with the desired number of sperm for artificial insemination. Immunological methods have emerged as an attractive alternative to flow cytometry and proteomic knowledge of X- and Y-sperm could be useful to the development of a new method. In this review, we identify the main applications of sexed semen, describe the existing methods and highlight future research opportunities in the field. We consider that immunological methods, based on sperm cell's surface proteins differentially expressed between X- and Y-sperm, could be an interesting and promising approach to semen sexing.

Differential proteins associated with plasma membrane in X- and/or Y-chromosome bearing spermatozoa in indicus cattle.

The differential proteins associated with plasma membrane of spermatozoa are less known, identification of which shall help overcome limitations of currently used methods of sperm sexing, considered as a high priority for livestock sector of many countries. This study has reported plasma membrane proteomics of unsorted spermatozoa and differential expression of plasma membrane-associated proteins between X- and Y-chromosome bearing spermatozoa of indicus cattle (Bos indicus). Isolation of plasma membrane fraction using percoll gradient, relatively a rapid method, from bovine spermatozoa has been reported to enrich isolation of plasma membrane proteins. Significant enrichment for plasma membrane-associated proteins was observed in plasma membrane fraction (p < .05) as compared to the total cell lysate using LC-MS/MS. Furthermore, these experiments were conducted in flow cytometry sorted, sexed-semen samples. Thirteen proteins were identified as differentially abundant between X- and Y-sorted spermatozoa. Among these, two proteins were downregulated in Y-sorted spermatozoa compared to the X-sorted spermatozoa (p < .05), while four and seven proteins could be noted in X- and Y-sorted spermatozoa, respectively. Proteins that are presumed to support sperm capacitation and sperm migration velocity were found to be abundant in Y-sorted spermatozoa while those associated with structural molecule activity were identified as abundant in X-sorted spermatozoa in the present study. Our study provides better insight into the plasma membrane proteomics of spermatozoa of indicus cattle and furnishes data that might aid in design and development of alternate and open technology for sex-sorting of semen.

Genetic consequences of terminal crossbreeding, genomic test, sexed semen, and beef semen in dairy herds.

The development of breeding tools, such as genomic selection and sexed semen, has progressed rapidly in dairy cattle breeding during the past decades. In combination with beef semen, these tools are adopted increasingly at herd level. Dairy crossbreeding is emerging, but the economic and genetic consequences of combining it with the other breeding tools are relatively unknown. We investigated 5 different sexed semen schemes where 0, 50, and 90% of the heifers; 50% of the heifers + 25% of the first-parity cows; and 90% of the heifers + 45% of the first-parity cows were bred to sexed semen. The 5 schemes were combined in scenarios managing pure-breeding or terminal crossbreeding, including genomic testing of all newborn heifers or no testing, and keeping Swedish Red or Swedish Holstein as an initial breed. Thus, 40 scenarios were simulated, combining 2 stochastic simulation models: SimHerd Crossbred (operational returns) and ADAM (genetic returns). The sum of operational and genetic returns equaled the total economic return. Beef semen was used in all scenarios to limit the surplus of replacement heifers. Terminal crossbreeding implied having a nucleus of purebred females, where some were inseminated with semen of the opposite breed. The F1 crossbred females were inseminated with beef semen. The reproductive performance played a role in improving the benefit of any of the tools. The most considerable total economic returns were achieved when all 4 breeding tools were combined. For Swedish Holstein, the highest total economic return compared with a pure-breeding scenario, without sexed semen and genomic test, was achieved when 90% sexed semen was used in heifers and 45% sexed semen was used for first-parity cows combined with genomic test and crossbreeding (+€58, 33% crossbreds in the herd). The highest total economic return for Swedish Red compared with a pure-breeding scenario, without sexed semen and genomic test, was achieved when 90% sexed semen was used in heifers combined with genomic test and crossbreeding (+€94, 46% crossbreds in the herd). Terminal crossbreeding resulted in lower genetic returns across the herd compared with the corresponding pure-breeding scenarios but was compensated by a higher operational return.

Production of sexed bovine embryos in vitro can be improved by selection of sperm treatment and co-culture system.

The study investigated the effects of sperm sorting, capacitation treatment and co-cultivation on sexed bovine in vitro embryo production. The effect of treatment and co-culture on production of embryos of the preferred sex from unsorted sperm was also studied. Sperm from five breeding bulls was used for fertilization of mature oocytes as follows: Experiment 1, sorted and unsorted sperm (bulls A-E) treated only with heparin in standard co-cultures; Experiment 2, sorted sperm (bulls A-E) treated with heparin-PHE (penicillamine, hypotaurine, and epinephrine) or heparin-caffeine in drop co-cultures; and Experiment 3, unsorted sperm (bull E) treated with either heparin-PHE or heparin-caffeine in both standard and drop co-cultures. In all bulls, treatment with heparin resulted in significantly (p < .05) reduced cleavage and blastocyst rates from sorted sperm, as compared with those from unsorted sperm. In bulls A, B, D and E, treatment of sorted sperm with heparin-PHE in drops significantly increased the blastocyst rate (p < .05). In unsorted sperm of bull E, heparin-PHE treatment in drops resulted in the XX/XY sex ratio inverse to that obtained by heparin-caffeine treatment in standard co-cultures (32.3%/67.7% and 66.7%/33.3%, respectively). In conclusion, the treatment of sorted sperm with heparin-PHE in modified drop co-cultures can be recommended for production of in vitro sexed embryos. The use of unsorted sperm for production of embryos of the preferred sex by selected capacitation treatment and co-culture can be the method of choice in bulls with low IVF yields from sorted sperm.

Spermatological parameters of immunologically sexed bull semen assessed by imaging flow cytometry, and dairy farm trial.

This study compared the quality parameters of bull semen sexed using an immunological method with those of conventional semen by imaging flow cytometry and applied this semen in dairy farm trials. Semen samples were collected from ten ejaculates from five bulls. Each sample was divided into two treatments: conventional semen (CON) and semen sexed using monoclonal male-specific antibodies combined with the complement system for cytotoxicity reaction (IC-sexed). After obtaining frozen-thawed semen, we used imaging flow cytometry to assess acrosome integrity, sperm sex ratio and viability. Sperm morphology was evaluated using eosin-nigrosin staining. The percentage acrosome integrity did not differ between IC-sexed and CON semen (P = 0.313). The sperm sex ratio showed that the percentage of live X-chromosome-bearing sperm was higher than that of live Y-chromosome-bearing sperm in IC-sexed semen (P = 0.001). IC-sexed semen showed a higher percentage of head and tail defects than did CON semen (P = 0.019). In field trials, 585 cows were subjected randomly to AI with CON or IC-sexed semen. The pregnancy rate of the IC-sexed group did not differ from that of the CON group (P = 0.535). However, IC-sexed semen produced a significantly higher percentage of female calves than did CON semen (P = 0.031). Thus, immunological sexing did not adversely affect the acrosome integrity of sperm. Furthermore, a female calf birth rate of over 74 % can potentially be achieved using IC-sexed semen. These findings could help farmers to replace heifers in their herds.

Small ruminant SexedULTRA™ sperm sex-sorting: Status report and recent developments.

Flow cytometry sperm sex-sorting based on the relative DNA difference between X- and Y-chromosome bearing populations is an established method that has allowed the production of pre-sexed offspring in a multitude of species and has been a commercial success in cattle production for the past twenty years. Several improvements to the technology and the processing methods have increased the sorting efficiency of ejaculates and the post-thaw quality of sex-sorted sperm, allowing for the fertility gap between conventional (non-sorted) and SexedULTRA™ sex-sorted sperm to be bridged. Small ruminant industries are now progressively testing sex-sorted sperm for application in their specific niches and environments. A review of the key advances and the recent developments in caprine, ovine and cervine sperm sex-sorting technology are described in this publication.

Effects of herd fertility on the economics of sexed semen in a high-producing, pasture-based dairy production system.

This study used a stochastic simulation model to estimate the potential economic benefit of using sexed semen in heifers only and in heifers and lactating cows in a high-producing, pasture-based system under 3 fertility scenarios. Three breeding strategies were modeled: (1) only heifers inseminated with sexed semen and cows inseminated with conventional unsexed semen (SSH); (2) both heifers and cows inseminated with sexed semen (SSHC); and (3) a reference scenario in which all females were inseminated with conventional, unsexed semen (CONV). Each scenario was evaluated under 3 herd fertility states: high (HF), medium (MF), and low (LF), which, under the reference scenario, corresponded to herd replacement rates of 21, 25, and 31%, respectively. The model estimated the economic profit, including the net present value of the genetic gain from selection intensity. The economic return from adoption of sexed semen strategies declined, with reduced levels of baseline herd fertility turning negative in the LF state. The mean (±SD) sexed semen advantage (SSA) per cow for HF-SSH, MF-SSH, and LF-SSH scenarios were €30.61 ± 8.98, €27.45 ± 7.19, and €14.69 ± 11.06, respectively. However, the SSA per cow for HF-SSHC, MF-SSHC, and LF-SSHC scenarios were €49.14 ± 15.43, €18.46 ± 30.08, and -€19.30 ± 57.11. The range in economic profit for SSA for SSH was most sensitive to calf prices in HF-SSH and the pregnancy rate of sexed semen as a percentage of conventional unsorted semen in MF-SSH and LF-SSH. The range in economic profit for SSA for SSHC scenarios was most sensitive to the pregnancy rate of sexed semen as a percentage of conventional unsorted semen in HF-SSHC, MF-SSHC, and LF-SSHC. This study highlights the effect of baseline herd fertility state on the financial advantage of adopting sexed semen in a pasture-based dairy production system.

Pre-synchronization of ovulation timing and delayed fixed-time artificial insemination increases pregnancy rates when sex-sorted semen is used for insemination of heifers.

This study was conducted to determine effects of pre-synchronization of ovulation timing among heifers and delayed fixed-time artificial insemination (TAI) with sex-sorted semen on proportion of heifers pregnant after TAI (PR/AI). Heifers were assigned to one of eight treatments: 1 and 2), 7-d CO-Synch + CIDR treatment regimen with administration of gonadotropin-releasing hormone and a CIDR insert on Day 0, prostaglandin F2α (PGF) at CIDR removal on Day 7, and TAI occurring 54 h later with conventionally processed (CTRL54-CNV) or sex-sorted semen (CTRL54-SEX); 3 and 4), same as CTRL54 but TAI delayed to 72 h with conventionally processed (CTRL72-CNV) or sex-sorted semen (CTRL72-SEX); 5 and 6), same as CTRL54 but additional administration of PGF on Day -7 and TAI with conventionally processed (PRE54-CNV) or sex-sorted semen (PRE54-SEX); 7 and 8), same as PRE54 treatments but TAI delayed to 72 h with conventionally processed (PRE72-CNV) or sex-sorted semen (PRE72-SEX). Proportion of heifers pregnant after TAI was greater (P ≤  0.02) with conventionally processed semen compared with sex-sorted semen, yet PR/AI did not differ (P =  0.14) between heifers in PRE72-CNV and PRE72-SEX groups. There were greater PR/AI in the PRE72-SEX (P =  0.03) than CTRL54-SEX group (46.1 % and 36.9 %) and there was no difference (P =  0.31) in PR/AI between CTRL54-CNV and PRE72-SEX groups (50.4 % and 46.1 %). In conclusion, pre-synchronization of ovulation timing among heifers combined with delayed TAI resulted in increased PR/AI with sex-sorted semen compared with the 7-d CO-Synch+CIDR treatment regimen.