Non-sorted Semen Research Articles

Fertility of conventional and of sex-sorted stallion sperm using SexedULTRA™ Genesis III technology

Sex-sorted (SS) bull sperm has been adopted worldwide due to the economic benefits and acceptable fertility (Crites BR et. al Theriogenology 118:126-129, 2018). Previous (15-20 years ago) studies using SS stallion sperm resulted in lower fertility when mares were bred with SS compared to non-sorted (NS) stallion sperm whether fresh (Buchanan BR Theriogenology 53:1309-1321, 2000); cool-stored (Lindsey et al. An. Reprod. Sc.85:125-130 2005), or frozen/thawed SS semen (Clulow et al. An. Reprod. Sc. 108:287-297, 2008). Recently, STgenetics, Navasota, TX, USA, a company dedicated to commercial SS semen, has developed a sperm sorter technology (SexedULTRA™ Genesis III) that reduces sorting time, increases the speed and efficiency of SS and reduces the amount of sperm damage (Gonzalez-Marin C. et al Theriogenology 114:40-45, 2018). In the present study, sperm quality (total motility, viability, and DNA damage) and fertility of mares bred with 40 million non-sorted (NS) or sex-sorted (SS) sperm were compared. Semen from 3 sexually active fertile stallions was diluted 1:3-1:4 in BotuSemen Gold supplemented with 0.06 mg/ml of Hoechst 33342 stain and cooled to 8°C for 18 h. Semen was then warmed to room temperature and centrifuged through a colloid suspension (EquiPure). The sperm pellet was resuspended to 250-300 million/ml using a proprietary chemically defined medium (ST Equiship,STgenetics, Navasota, TX) and transported at ∼20°C for approximately 45 min to the sorting facility. When 40 million SS were harvested (approx 2-3h) SS or NS semen [LCC1] [LCC2] were loaded in 4-5 0.5 mL straws and inseminated (1x/cycle) using a deep-horn artificial insemination technique. A total of 17, 8-21 year old mares were bred on 65 cycles. When mares had uterine edema of 3/5 and a dominant follicle of > 35 mm, 1 mg of deslorelin acetate (Sucromate®) was administered IM. Mares were inseminated ∼24h after deslorelin administration and examined for ovulation 12-18h post AI. Seven to 8d post-ovulation non-surgical embryo recovery was performed. Total sperm motility (86%) and viability (84%) were higher and DNA damage was lower (4%) in SS compared to NS sperm (78, 82,12%) (p<0.05). Embryo recovery rates were similar for NS (46.6%) compared to SS sperm (40%) (p>0.05). The expected sex of all embryos was correctly identified by PCR using the presence or absence of the SRY gene (Choi YH et al, Reproduction 140: 893 – 902, 2010). Overall, sperm quality of SS sperm was similar or higher to that of NS stallion sperm while embryo recovery rates were similar. Further studies are underway to validate this technology on a commercial scale.

The Difference in Subpopulation Structures in Sex-Sorted and Non-Sorted Semen by Discriminant Analysis of Bull Sperm Motility Data Collected by a Computer-Assisted Sperm Analysis System

We examined the motility characteristics of sperm from sex-sorted bull semen as a model for low-quality (expected low conception rate) sperm compared to that of sperm from non-sorted semen (expected average conception rate) using cluster analysis followed by discriminant analysis. The results indicated that sex-sorted semen contained sperm with hyperactivation-like motility as the main subpopulation immediately after thawing, and this subpopulation decreased after 2 h of incubation. The main subpopulation in the non-sorted semen had progressive motility that was maintained during incubation. A conventional comparison of the mean values of kinematic parameters could not distinguish the samples. In conclusion, discriminant analysis using data from a cluster analysis of motile sperm may accurately describe differences in the structures of sperm motility subpopulations between low and normal fertility semen.

173 Pregnancy rates in suckled beef cows synchronised with progesterone/oestradiol-based protocol and inseminated with conventional or sexed-sorted semen

An experiment was designed to evaluate pregnancy rates per AI (P/AI) in suckled beef cows synchronised with a progesterone (P4)/oestradiol-based protocol and AI with non-sexed (conventional) semen and with a sexed-sorted semen with 65% X-bearing sperm and 35% Y-bearing sperm that was named SuperConventionalTM. Angus and Hereford suckled cows (n=558), 60-90 days postpartum, with a body condition score of 2 to 3.5 (1-to-5 scale) and 48% (269/558) with a corpus luteum, were randomly allocated to be AI with non-sexed (conventional) or SuperConventional semen. The experiment was performed on 3 farms using straws with sexed-sorted SuperConventional semen containing 6 or 8 million sperm and non-sorted conventional semen with 25 million sperm per straw. Semen was from split ejaculates from two Angus bulls. All cows received a P4 device with 0.6g of P4 (Pluselar, Calier) and 2mg of oestradiol benzoate (Calier) on Day 0. All P4 devices were removed on Day 8, and all cows received 400IU of equine chorionic gonadotrophin (Vetegon, Calier), 150µg of d+cloprostenol (Veteglan, Calier), and 1mg of oestradiol cipionate (Calier) at the same time. All cows were also tail-painted and observed for signs of oestrus. Cows with &amp;gt;30% of the tail paint rubbed off by 48-50h after P4 device removal were AI at that time with either SuperConventional or conventional semen. Those not showing oestrus by 48-50h received 10µg of buserelin (Pluserelina, Calier) at that time and were AI with either SuperConventional or conventional semen 50-52h after P4 device removal. Pregnancy was diagnosed using ultrasonography 60 days after AI, and all pregnancies were sexed to determine the proportion of female pregnancies. Data were analysed using GLM for binary data. The P/AI were different between cows showing or not showing oestrus (P&amp;lt;0.01) but did not differ among the three types of semen used (Table 1). The percentage of cows pregnant with female fetuses was 51% (50/98) for cows AI with conventional semen and 65.5% (55/84) and 65.3% (51/78) for those AI with SuperConventional semen with 8 million or 6 million sperm per straw, respectively. In conclusion, sexed-sorted semen with 65% of X-bearing sperm and 6 or 8 million sperm per straw can be successfully used to inseminate suckled beef cows. Furthermore, results confirm those of previous studies that cows in oestrus at the time of fixed-time AI have higher pregnancy rates than cows not in oestrus in P4/oestradiol-based synchronisation programmes. Table 1.Effect of semen type and oestrus expression on pregnancy rates per AI in suckled beef cows synchronised with a progesterone/oestradiol-based protocol Semen type In oestrus,% (n) Not in oestrus,% (n) Total,% (n) Conventional (25 million sperm) 59.8 (82/137)a 32.0 (16/50)b 52.4 (98/187) SuperConventional (8 million sperm) 53.7 (73/136)a 20.0 (10/50)b 44.6 (83/186) SuperConventional (6 million sperm) 50.4 (63/125)a 26.7 (16/60)b 42.7 (79/185) Total 54.8 (218/398)a 26.3 (42/160)b a,bDifferent superscripts denote differences in pregnancy rates per AI between cows in oestrus and not in oestrus by the time of fixed-time AI (P&amp;lt;0.01). Research was supported by Fondo Nacional de Ciencia y Tecnología (FONCYT PICT 2017-4550) and UNVM.

Economic evaluation of artificial insemination of sex-sorted semen on a Brown Swiss dairy farm-A case study.

Artificial insemination using sex‐sorted semen is employed to efficiently increase the number of female dairy calves born. Previous studies have determined that using sex‐sorted semen is beneficial to improve the management, but the mechanism by which it increases cattle numbers through objective indices of breeding remains unclear. This study focused on a Brown Swiss cattle herd in which frozen female sex‐sorted semen was systematically employed to increase the number of cattle. We analyzed the correlation between the increase in the number of cattle and the screening accuracy of sex‐sorted semen, measuring indices such as pregnancy rate and birth rate of female calves. Study revealed that: (1) production cost for female calves is influenced by the pregnancy rate, rate of female calves, and using sex‐sorted semen is less expensive than using nonsorted semen; (2) improvements in screening accuracy nearly doubled the number of cows and tripled the number of heifers in 5 years; and (3) use of sex‐sorted semen improved milk quality. The pregnancy rate was lower when sex‐sorted semen was used, but the birth rate of heifers was improved. Results suggest that artificial insemination using sex‐sorted semen is beneficial because it economically produces offspring to increase the herd.

In this issue – September 2017

In this issue – September 2017

Sex-sorted sperm in farm animals: scale of use, the effectiveness of insemination, risk factors

The aim of the study was to present the scale of use, risk factors and possibilities, which sorter semen gives in biotechnics used in reproduction of cattle. Modern sorters allow for the evaluation of 6 million X and Y spermatozoa per hour. Sex-sorted semen, which is commercially used, contains 2.1 x 106 of spermatozoa. It is used mostly in AI of milk heifers, mainly in large cattle herds. Sorted semen containing Y spermatozoa is sold less often in the world than the one with X spermatozoa. The percentage of the desired sex of the young is higher than 90. The pregnancy rate after application of sorted semen is about 20–25% lower than after insemination of non-sorted semen and depends on a number of factors. The main factors are: breed of female, service number, the herd of origin, the depth of semen deposition, the bull producing semen, ambient temperature and technical parameters during sperm sorting. A number of methods have been developed to improve conception rate, including timed artificial insemination (TAI) and synchronization of heat and ovulation. Results of donor inseminations with the use of sorter semen are presented, with the lower percentage of embryos suitable for the transfer and embryos of the highest quality highlighted. Previous studies do not indicate a reduction of the conception rate after the transfer of embryos obtained in vitro and in vivo after fertilization using sorted semen. It remains difficult to justify a significant increase in the frequency of stillbirths of bulls after using sorted sperm. Similarly, 16% of stillbirths of bulls were observed after embryo transfer, when donors were inseminated with sorter semen. The percentage of stillbirths of bulls after embryo transfer with the use of conventional semen is 9%. The sorted semen is not often used for inseminations in pigs, sheep and goats.

Fertilization rate and embryo production of superovulated dairy cows after insemination with non-sorted and sex-sorted semen

The aim of this study was to evaluate the fertilization rate of cows that were superovulated and artificially inseminated with sex-sorted semen. Cows were treated with an intravaginal progesterone device plus estradiol benzoate (day 0). Superstimulation treatments began four days after with eight applications of FSH at 12 h intervals. D-Cloprostenol was administered on day 6. Progesterone device was removed on day 7, and LH was administered on day 8. The treatments were divided as follows: NonSx, two AI with non-sorted semen were conducted 12 and 24 h after LH; Sx12&24, two AI with sex-sorted semen were conducted 12 and 24 h after LH; and Sx24&36, two AI with sex-sorted semen were conducted 24 and 36 h after LH. Embryos were recovered on day 16 and were evaluated and classified. Percentage of fertilized embryos tended to be greater for the non-sorted semen than the sex-sorted semen. The number of unfertilized oocytes was smaller when the non-sorted semen was used relative to the sex-sorted semen. There was no difference between the treatments that used sexed semen. In conclusion, the use of sex-sorted semen in superovulated dairy cows results in greater numbers of unfertilized oocytes than non-sorted. However, when only sorted semen is used AI should be performed 24 and 36 h after LH.

215 PRODUCTION OF PROGENY WITH SEXED WHITE HANWOO (KOREAN NATIVE ALBINO CATTLE) SEMEN

The aim of the study was to produce sexed progeny of the genetically confined White Hanwoo (albinism). A White Hanwoo bull was selected as the semen donor. The X-bearing sperm were sorted by MoFlo XDP cell sorter from Korea Sexing Biotech (Daegu, South Korea). To compare pregnancy and birth rates as well as semen quality, a Korean proven bull (KPN) was used as control. Total number of nonsorted sperm was 20 × 106 per straw. Double insemination of 2 or 4 × 106 sexed and frozen X-bearing sperm were performed on Hanwoo heifers. The abnormality of nonsorted albino spermatozoa (5 ejaculates) was 24.9 ± 7.31% (but 18.7 ± 6.2% C in KPN 768, derived from 5 straws) with a higher number of distal reflex abnormality in midpiece (11.7% compared with that of KPN 768, 5.6%). There were no differences in pregnancy and birth rates when 2 or 4 × 106 X-bearing sperm were inseminated but nonsorted semen had higher pregnancy rates (P &lt; 0.05). The pregnancy rates of KPN 768, 2 × 106 cells, and 4 × 106 cells X-bearing sperm of White Hanwoo cattle were 85.0% (17/20), 26.3% (5/19), and 50% (7/14), respectively. The birth rates were 80.0% (16/20), 15.8% (3/19), and 21.4% (3/14), respectively. The female offspring rates were 43.8% (7/16), 100% (3/3), and 100% (3/3) (P &lt; 0.05). These results indicated that sex-sorted albino Hanwoo semen could be used for the production of wanted progeny with 2 × 106 cells/straw for AI to heifers.

Incorporation of sexed semen into reproductive management of cow–calf operations

The objective of this experiment was to compare reproductive performance and weaning outcomes of beef cows inseminated with sexed or conventional semen. Over 2 consecutive years, lactating Angus×Hereford cows were assigned to an estrus synchronization+artificial insemination (AI) protocol. At the time of AI, cows were ranked by parity and assigned to be inseminated with conventional non-sorted semen (CONV; n=454) or with semen sorted for male sperm (SEXED; n=439). Beginning 18 days after AI, cows from both treatments were grouped and exposed to mature bulls for 50 days (1:25 bull to cow ratio). Cow pregnancy status to AI was verified by detecting a fetus via transrectal ultrasonography 40 days after AI. Calf birth date, sex, and birth BW were recorded during the subsequent calving season. Cows that were diagnosed as pregnant during the transrectal ultrasonography exam and gave birth during the initial 2 weeks of the calving season were considered pregnant to AI. Pregnancy rates to AI and final pregnancy rates (AI+bull breeding) were reduced (P≤0.05) in SEXED compared with CONV cows. The proportion of male calves born to AI or AI+bull breeding was greater (P<0.01) in SEXED compared with CONV cows. No treatment effect was detected (P=0.34) for weaning rate, whereas SEXED cows had a greater (P<0.01) proportion of steers in the weaned calf crop compared with CONV cows. Steers and heifers from SEXED cows were younger (P<0.01), whereas only SEXED heifers were lighter (P=0.05) at weaning compared with cohorts from CONV cows. Across genders, calves from SEXED cows had reduced (P≤0.01) weaning age and BW compared with calves from CONV cows. Cows assigned to SEXED had greater (P=0.05) kg of steer weaned/cow exposed to breeding, but reduced kg of heifer weaned/cow exposed to breeding (P<0.01) compared with CONV cows. Across genders, SEXED cows tended (P=0.09) to have reduced kg of calf weaned/cow exposed to breeding compared with CONV cows. In summary, inseminating beef cows with sexed semen reduced pregnancy rates, but increased the proportion of steers weaned and kg of steers weaned/cow exposed to breeding. However, overall kg of calf weaned/cow exposed to breeding was not improved by the use of sexed semen, particularly because of its negative impacts on weaning age and BW of the heifer progeny.

Pregnancy rates of lactating cows after transfer of in vitro produced embryos using X-sorted sperm

The main objective was to determine the efficacy of using X-sorted sperm to produce embryos in vitro for transfer into lactating dairy cows. Cows were bred by timed artificial insemination (TAI) using nonsorted semen or X-sorted sperm, or they received a fresh embryo produced in vitro by fertilization with X-sorted or nonsorted sperm using timed embryo transfer (TET). Pregnancy rates at approximately Day 32 averaged over all dairies were 39.3 ± 3.2% (least-squares mean ± SEM) for TAI nonsorted, 27.3 ± 3.4% for TET nonsorted fresh embryos, and 30.2 ± 3.3% for TET X-sorted fresh embryos (TAI vs. both TET groups, P < 0.05; 206 to 233 cows per group). Pregnancy losses between approximately Day 32 and term ranged from 16% to 37%, the latter from TET with X-sorted sperm. Pregnancy losses to term were higher for cows receiving embryos produced in vitro than for cows bred by TAI. Calves produced via TET were not substantively different from AI controls in physical measurements or standard blood chemistry profiles.

368 CONCEPTION RATE AT FIRST ARTIFICIAL INSEMINATION WITH SEXED SEMEN OR CONVENTIONAL SEMEN IN CROSSBRED DAIRY COWS

The use of sexed semen in dairy cattle almost eliminates the birth of unwanted male calves and can lead to increased milk production by means of genetic selection exerted on the females. However, AI with sexed semen still presents several obstacles to overcome. Holstein-Zebu cows are responsible for 80% of the milk produced in Brazil. These crossbred animals are used extensively throughout the country because their physiologic and morphologic characteristics are compatible with milk production in the tropics which results in an economic performance advantage. The objective of this study is to evaluate the conception rates with sexed semen, comparing it to the use of non-sorted semen in crossbred dairy cows. In addition, two insemination times had been studied to determine the best timing for AI with the sexed semen. Both studies were carried out in the Experimental Station Seropedica of Pesagro-RJ, located in the state of Rio de Janeiro, Brazil. The experiment was done in 2008/2009 (January to April). In the first Trial 56 Holstein-Zebu females, composed of 1/2 to 7/8 Holstein genetics, were inseminated 12 h after estrus detection with non-sorted semen (control group n = 29, 14 heifers and 15 cows) or with sexed semen (experimental group n = 27, 13 heifers and 14 cows). The semen utilized was from the same bull and thawing was performed intravaginally. In the second trial, 55 females were inseminated only with sexed semen from the same bull, with thawing in water at 35°C, at two different times: 12 h after estrus observation (control group n = 27, 12 heifers, 4 primiparous, and 11 multiparous) and 15 h after estrus observation (experimental group n = 28, 12 heifers, 5 primiparous, and 11 multiparous). The pregnancy diagnoses were done by rectal palpation and transrectal CAT scan at Day 45. Statistical analyses had been done using Chi-square. The conception rate in the first trial was of 17.2% (5/29) for sexed semen and 53.8% (14/26) for non-sorted semen (P &lt; 0.05). In the second trial, the conception rate was of 37.0% (10/27) for insemination with sexed semen, 12 h after estrus observation and 50.0% (14/28) for the females inseminated 15 h after estrus observation (P &gt; 0.05). It is possible that one of the causes of fertility reduction with sexed semen is that sexed-sperm remain viable for a shorter time after the insemination. Inseminations with sexed semen done 15 h after estrus detection, which can be more synchronized with the time of ovulation, and when accompanied by closely controlling the thawing process, show results similar to those obtained with conventional semen.

363 ENHANCEMENT OF COMMERCIAL BOVINE IN VITRO EMBRYO PRODUCTION BY SEXING FROZEN SEMEN PRIOR TO INSEMINATION

The high commercial demand on production of genetically valuable female cattle was emphasized after use of sex-sorted semen in commercial in vitro embryo production (IVEP) programs and a reduction of interest in the use of embryo biopsy followed by PCR. Brazil is a leader in IVEP of bovine embryos, and frozen-thawed, sex-sorted samples become a new option for embryo sexing. There are only a few reports in the literature using this new technique for IVEP. Most reports have involved the use of frozen-thawed then sorted for use with AI in Europe. In the present study, frozen semen from three Nelore bulls was used and the blastocyst production rate was evaluated at Day 7 of IVP and compared to previous data from the same bulls and a batch of non-sorted semen. Cumulus oocyte complexes obtained from high genetic merit donors by ovum pickup were matured (TCM-199, supplemented with FCS, LH, FSH, estradiol, pyruvate, and antibiotics) for 24 h and fertilized (Fert-TALP supplemented with BSA, phenylalanine, and heparin) for 18-22 h (Day 0) in vitro. Frozen semen straws were thawed at 35°C for 30 s in a water bath and then selected by centrifugation at 800 g on discontinuous Percoll™ gradients (45 :90%). Samples of frozen-thawed sex-sorted semen were processed from straws from bulls that in some cases may already be dead. Frequently, 3 to 5 straws were thawed, flow-sorted, and then centrifuged to provide sufficient sperm to fertilize 100 oocytes. On Day 1, presumptive zygotes were transferred to culture media (SOFaa supplemented with BSA and FCS) and on Day 7 blastocyst production rate was evaluated. The present results were commercially satisfactory because the frozen-thawed sex-sorted semen from all three bulls performed comparably and, in one case, better than non-sorted semen from the same bulls. Therefore, its use is extremely attractive in commercial bovine IVEP systems and is a more efficient system than embryo sexing. Table 1.Embryo production rates at 7 days post insemination

375 PERFORMANCE OF DIFFERENT BOVINE BREEDS ON IN VITRO COMMERCIAL EMBRYO PRODUCTION USING SEX-SORTED SEMEN

Commercial bovine in vitro production (IVP) systems using sex-sorted semen, instead of the invasive technique involving embryo biopsy followed by PCR, have a superior influence on embryo production rates. However, due to damage from flow cytometric procedures and cryopreservation, blastocyst rates using sex-sorted semen remain lower than from conventional non-sorted semen. The aim of this study was to describe embryo production rates using sex-sorted semen during the past year in different bovine breeds. Cumulus oocyte complexes obtained by ovum pickup (OPU) from high genetic merit donor cows were matured (TCM-199, supplemented with FCS, LH, FSH, estradiol, pyruvate, and antibiotics) for 24 h and fertilized (Fert-TALP supplemented with BSA, phenylalanine, and heparin) for 18-22 h (Day 0) in vitro. Frozen sex-sorted semen samples from different bulls were thawed at 35°C for 30 s in a water bath and then selected by centrifugation at 800 g on discontinuous Percoll™ gradients (45 : 90%). Presumptive zygotes were transferred to culture medium (SOFaa supplemented with BSA and FCS) on Day 1 and blastocyst rate was evaluated on Day 7. During the past year, sex-sorted semen was used more frequently on Bos taurus (Holstein), Bos indicus (Gyr), and crossbred (Gyrolando) milk cattle than in meat breeds (Nelore, Guzera, and Brahman). The large numbers of oocytes routinely recovered in Nelore cows could be attributed to the female valorization in the market due to high potential in new generations and repositioning of genetic value. The effect of subspecies (Bos taurus taurus and Bos taurus indicus) could be also observed in blastocyst rate, with better results than Holstein, showing the same tendency when conventional (non-sexed) semen is used. Embryo production in Brahman and Nelore with sex-sorted semen was lower than with conventional (non-sexed) semen, probably due to damage to the sperm during the sorting process, thereby changing patterns of fertilization, involving the acrosome reaction, for example. This points to the need for more studies on cellular integrity during the sorting process as well improvement of IVP procedures with sex-sorted semen. Table 1.Embryo production rates according to different bovine breeds

Embryo production after in vitro fertilization with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm

The objective of this study was to determine the in vitro fertilizing capacity of bull sperm derived from fresh or frozen samples and subjected to sex sorting and re-cryopreservation. Four sperm types were assessed for their ability to fertilize and sustain early embryo development in vitro. Semen from three Bos taurus bulls of different breeds (Jersey, Holstein and Simmental) was collected and either sorted immediately and then frozen (SF) or frozen for later sorting. Frozen sperm destined for sorting were thawed, sex-sorted, and re-frozen (FSF) or thawed, sex-sorted (FS), and used immediately for in vitro fertilization (IVF). Frozen-thawed nonsorted semen from the same ejaculate was used as a control. Oocytes from donor cows were aspirated via ovum pick-up and matured in vitro prior to IVF and culture. On average, 19.0 ± 1.7 (mean ± SEM) oocytes were aspirated per donor cow, of which 74.4 ± 2.2% were selected for maturation. The proportion of cleaved embryos (Day 3) did not differ between sperm groups (P = 0.91). Likewise, IVF with FSF sperm resulted in similar Day 7 blastocyst rates (as a percentage of total oocytes) as those of control, SF, and FS sperm (FSF, 34.5 ± 4.7; control, 32.2 ± 4.6; SF, 35.9 ± 4.8; and FS, 26.9 ± 4.1%; P = 0.23). These encouraging results show that frozen-thawed sex-sorted sperm may be re-frozen and used for in vitro embryo production with similar blastocyst production as that of nonsorted frozen-thawed and sex-sorted frozen-thawed sperm.

267 DIFFERENCES IN BULL SPERM NUCLEAR SHAPE BETWEEN FLOW-SORTED AND NON-SORTED SPERM USING FOURIER HARMONIC ANALYSIS

The objective of the study was to evaluate sperm nuclear shape in sex-sorted and non-sorted semen using Fourier Harmonic Amplitudes (FHA). Frozen semen was obtained from a single commercially available source. Mature bulls (n = 15) from the same breed with an average age of 4.42 ± 2.08 years were collected and the semen was either frozen or X-chromosome sorted using a flow cytometer and then frozen. Frozen straws were transported to the lab and analyzed for FHA. Briefly, straws were thawed and cells were incubated with 1.6 μm Hoechst 33342. Cells were then washed, fixed, dried to a slide and analyzed for nuclear head shape. Harmonic amplitudes 0 to 5 (HA0–HA5), derived from FHA, were previously shown to be an accurate, objective, and repeatable measure of sperm nuclear shape. HA0 describes the overall nuclear size of the sperm, whereas HA1 describes the anterior head, HA2 the length of the sperm along the longitudinal axis, and HA3 to 5 the distal, post-nuclear curvature of the sperm head. Each unit of semen was evaluated for motility and FHA. There was a significant decrease in motility in the sorted group (77 ± 1% v. 54 ± 3%; P &lt; 0.0001). Multivariate ANOVA showed that there were differences between the sorted and non-sorted groups in HA1 to 4 (P &lt; 0.02). Harmonic amplitude means ± SD (microns) for sorted and non-sorted treatments are as follows: HA1 (0.117 ± 0.003 v. 0.109 ± 0.003), HA2 (1.087 ± 0.005 v. 1.063 ± 0.005), HA3 (0.139 ± 0.003 v. 0.130 ± 0.003), and HA4 (0.201 ± 0.004 v. 0.191 ± 0.004), respectively. The nuclear shape of X-sorted sperm is longer and more pinched in both the anterior and posterior head. Interestingly there was no difference in HA0 (P = 0.119) indicating that the overall size of the sperm head is not affected by the sorting process. The differences in harmonic amplitudes may be due to the size and a restricted location of the X v. Y chromosome in the sperm nucleus.

Insemination with Sex Sorted Fresh Bovine Spermatozoa Processed in the Presence of Antioxidative Substances

Flow cytometrically sex sorted spermatozoa are reduced in their fertilizing capacity, particularly when stored either in cooling extender or after freezing in liquid nitrogen. So far, preservation methods for sorted spermatozoa have differed only marginally from procedures used for unsorted semen. In the present study, a TRIS extender was modified to balance major cell damage caused by the sorting process and by liquid storage of the sorted spermatozoa. The new extender, containing a combination of antioxidants (AO) and bovine serum albumin (BSA), significantly increased the lifespan and fertilizing capacity of sex sorted spermatozoa. No significant differences were observed between unsorted controls and sorted samples for motility and status of sperm membranes as tested by fluorescein-isothiocyanat-peanut agglutinin/propidium iodide (FITC-PNA/PI). Acrosome integrity of spermatozoa was significantly better when semen was stored at 15 degrees C for 24 and 48 h in an extender containing AO with or without BSA as compared with controls (p < 0.05). There were no significant differences, in pregnancy rates of heifers inseminated at a natural oestrus, between unsorted controls (16/24, 66.7%) and both sorted groups (AO + BSA: 18/31, 58.1% and AO-BSA: 12/22, 54.5%). Additionally, it was shown for the first time that artificial insemination (AI) with liquid sexed bull spermatozoa stored for 72 h after sorting can result in pregnancy rates similar to AI with non-sorted semen.

Fertility in heifers and cows after low dose insemination with sex-sorted and non-sorted sperm under field conditions

The present study was performed to test fertility after low dose insemination with sexed and non-sexed sperm in dairy cattle under field conditions in Switzerland. Spermatozoa were stained with Hoechst 33342 and sorted by flow cytometry. A total of 132 heifers and cows were inseminated with 2 × 10 6 X-bearing, frozen-thawed sperm (A) and 91 animals were inseminated with the same dose using non-sorted, frozen-thawed sperm (B). Pregnancy examination by ultrasound was performed twice, 30–40 days (PE1) and 70–90 days (PE2) after insemination. The pregnancy rates after PE1 were 33.3% (9/27) and 59.3% (16/27) in heifers ( P = 0.05) and 27.6% (29/105) and 28.1% (18/64) in cows ( P > 0.05) for groups A and B, respectively. Embryonic losses between PE1 and PE2 in heifers were 11.1% (1/9) and 0% (0/16) and in cows 17.2% (5/29) and 5.6% (1/18), the differences between groups A and B not being significant ( P > 0.05). Calving rates in heifers were 29.6% (8/27) and 57.8% (15/26), whereas in cows 22.1% (23/104) and 23.4% (16/63) gave birth to calves (for both groups P > 0.05). The sex ratio was different ( P < 0.05) between A (85.3%) and B (58.6%). From our results it can be concluded that conception rates of sorted and non-sorted semen are similar using an insemination dose of 2 × 10 6. Fertility may be increased by improving sexing technology and animal management.

287IN VITRO PRODUCTION OF HOLSTEIN EMBRYOS USING SEX SORTED SEMEN

Our objective was to explore the synergy between sexed semen and in vitro embryo production and assess benefits of these technologies on commercial farms. Ovaries were collected from high genetic merit Holstein cull cows via colpotomy or at the time of slaughter. Oocytes were aspirated from the ovaries, fertilized 20–24h later, and matured to the morula or blastocyst stage. Embryos were transferred into recipient Holstein cows and heifers on the same farms. Seven Wisconsin herds participated, and 365 embryos were produced from 104 donor cows. Only 272 of these embryos were transferred due to limited availability of recipients. Sexed semen from three Holstein sires was used. On average, 3.5±0.37 transferable embryos were produced per donor, including 1.4±0.18 grade 1 embryos and 1.5±0.20 grade 2 embryos. Individual farms averaged from 1.6 to 5.8 transferable embryos per donor. Laboratory data also revealed interesting results. On average 43.7±4.0 oocytes were collected per donor, and the number of usable oocytes (33.9±3.4), and percent embryos cleaved (52.1±1.9), were significant predicators of the number of blastocysts developed. We divided the usable oocytes and embryos cleaved per donor into quartiles. The fourth quartile for embryos cleaved was significantly greater (P&amp;lt;0.05) than the lower three quartiles, and the usable oocyte quartiles all significantly differed from each other. Semen freeze date was also a significant predicator of the number of blastocysts developed, suggesting significant variation in the quality of sorted semen per ejaculate. To preliminarily test the effect of sorting on the percentage of embryos developing to blastocyst stage, oocytes were recovered from ovaries collected at a slaughterhouse and fertilized using non-sorted semen or sex-sorted semen from the same sires. Oocytes (n=3312) fertilized using non-sorted semen tended (P=0.06) to produce more embryos developing to blastocyst stage than oocytes (n=1577) fertilized using sex-sorted semen (20.1±2.9% v. 12.2±2.3%, respectively). Preliminary pregnancy results show strong farm and sire effects. Overall conception rate was 36% for heifer recipients and 18milking cow recipients. These results suggest that low cost in vitro embryo production may have promise as an early system for utilizing sexed semen in dairy cattle breeding programs.