Non-sorted Sperm Research Articles

Is the proAKAP4 a suitable biomarker of X-sorted sperm quality from Nelore and Gir bulls?

ProAKAP4 is a protein precursor of AKAP4 present in the principal piece of the flagellum, associated with sperm motility. It has been described in several species, but its concentration has never been measured in sex-sorted sperm. Thus, the study aimed to test the proAKAP4 as a suitable sperm quality biomarker for Bos indicus X-sorted sperm. To achieve the study’s goal, 14 semen straws from different bulls were evaluated, six X-sorted and eight non-sorted. Sperm kinetics and morpho-functional evaluations were carried out, in addition to the concentration of proAKAP4. The characteristics evaluated were compared by the t-test and correlated by the Pearson’s correlation. A difference was found for total motility (P = 0.014), rapid sperm (P = 0.020), and sperm area (P = 0.013) between the non-sorted and X-sorted sperm. The proAKAP4 concentration was higher in the X-sorted sperm (X-sorted sperm: 67.54 ng/106 spermatozoa; non-sorted sperm: 29.76 ng/106 spermatozoa), but it was not correlated to the kinetics or morpho-functional characteristics evaluated. However, the proAKAP4 in non-sorted sperm showed a correlation to sperm total and progressive motility, rapid sperm, and sperm membrane integrity. The study concluded on the unreliability of the proAKAP4 as a biomarker to indicate sperm quality in the X-sorted sperm.

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.

Assessment of semen quality and anti-oxidative enzyme activity between bovine sex-sorted and non-sex-sorted frozen-thawed semen.

In the current study, the difference between the sex-sorted and non-sex-sorted frozen semen of Holstein Friesian breed cattle was investigated. Significant variation (p < .05) was found in the semen quality parameters such as motility; vitality; acrosome integrity rate; the anti-oxidative enzyme activity including GSH (glutathione); SOD (superoxide dismutase); CAT (catalase); GSH-Px (glutathione peroxidase) and the rate of fertilization. The results showed that the sperm acrosome integrity and motility of the non-sorted sperm were higher compared to sex-sorted sperm (p < .05). The linearity index and mean coefficient analysis revealed that the percentage of 'grade a' in sex-sorted sperm were significantly (p < .05) lower than non-sorted sperm. Interestingly, low SOD level and high CAT level was found in the non-sexed semen than in the sexed semen (p < .05). Furthermore, the GSH and GSH-Px activity in the sexed semen was found lower than the non-sexed semen (p < .05). In conclusion, sperm motility characteristics were lower in sex-sorted semen than in non-sex-sorted semen. This might be related to the complex process of sexed semen production, which could reduce sperm motility and movement characteristics, acrosomal integrity, CAT, SOD, GSH and GSH-Px, and finally lead to the decline in the fertilization rate.

The reproductive success of bovine sperm after sex-sorting: a meta-analysis

In the three decades since its inception, the sex-sorting technology has progressed significantly. However, field studies report conflicting findings regarding reproductive outcomes. Therefore, we conducted this meta-analysis of all trials published between 1999 and 2021. Non-return rates after 24 or 60 d (NRR 24/60), pregnancy, calving, abortion, and stillbirth rates were compared after AI with sex-sorted vs non-sorted sperm. Additionally, the impact of recent developments in the sex-sorting technology was assessed. Of 860 studies found, 45 studies (72 trials) provided extractable data and were included. Overall, the results of this meta-analysis provided evidence that the NRR 24/60 was diminished by 13%, pregnancy rates were reduced by 23% (25% cows, 21% heifers) and calving rates were reduced by 24% when using sex-sorted sperm. Enhancing the dosage to 4 million sex-sorted sperm/straw (including recent improvements, high vs low dose) as well as using fresh sex-sorted sperm (sorted vs non-sorted) increased pregnancy rate ratios by 7 percentage points. The refinement of the sex-sorting technology after 2015 resulted in a lowered reduction of pregnancy and calving rate of 19% and 23%, respectively. Whereas abortion rates were similar, the stillbirth of male calves was increased by 6.3%.

Comparison between in vitro embryo production using Y-sorted sperm and timed artificial insemination with non-sorted sperm to produce crossbred calves

Due to the increasing use of in vitro embryo production (IVEP) and the importance of crossbreeding for beef production, pregnancy rates of Nelore recipients were evaluated following Fixed Time Embryo Transfer with fresh or vitrified IVEP embryos produced with Y-sorted sperm of Angus bulls (B. taurus) or Fixed Time Artificial Insemination using non-sorted sperm. For IVEP in Experiment 1, oocytes were obtained using Ovum Pick Up (OPU) (n = 84 embryos) or from ovaries from a slaughterhouse (SLAUGHTER, n = 66 embryos). In Experiment 2, with oocytes obtained by OPU, IVEP embryos were fresh (FRESH, n = 271) or after vitrification/warming (VITRIFIED, n = 79) and PR was compared with FTAI (n = 239). In Experiment 1, cleavage rates were 63.8% and 39.1% for OPU and SLAUGHTER groups, respectively (P = 0.02), and blastocyst rates were 30.5% and 14.7%, respectively (P = 0.09). The PR was similar when considering the source of oocytes (OPU = 35.7%; SLAUGHTER = 25.8%; P = 0.17). In Experiment 2, there was no difference in PR for FRESH or VITRIFIED embryos (34.3% and 30.4%, respectively, P = 0.72), but lesser than FTAI (47.7, P = 0.002). It is concluded that the IVEP with Y-sorted sperm associated with vitrification or embryos produced with oocytes from different sources did not affect PR when there was transfer of crossbred embryos into recipients, and can optimize large-scale application of IVEP technology; however, FTAI pregnancy rates with non-sex sorted sperm were greater.

Effect of sorting boar spermatozoa by sex chromosomes on oviduct cell binding

This study examined the hypothesis that flow sorting sperm by sex chromosomes affects oviduct cell binding which would influence formation of the sperm reservoir in the oviduct. The sperm-rich fraction from boars (n = 5) was collected, sperm were stained with Hoechst 33342 and sorted. Sperm were sorted based on the presence of either an X or Y chromosome and placed into the following treatments: 1) sperm selected for the Y chromosome, 2) selected for the X, 3) an equal mixture of sorted X and Y, and 4) a control of non-sorted sperm from the same collection. Samples were tested for oviduct cell binding within 12 h of sorting. Additionally, sperm were analyzed for motility characteristics, acrosome status, and binding to the two oviduct glycan motifs that bind porcine sperm, biantennary 6-sialylated N-acetyllactosamine on a mannose core (bi-SiaLN) and sulfated LeX trisaccharide (suLeX). The disaccharide found within both glycan motifs, N-acetyllactosamine (LacNAc), was used as a control. Sperm binding to oviduct cells was reduced by more than half in the three sorted samples when compared to the control sperm that were not sorted. The percentage of sperm that were motile 24 h after sorting was also decreased significantly in each of the sorted sample groups when compared to the unsorted control. In contrast, sorting did not decrease the percentage of sperm that bound purified soluble glycans or the location on sperm to which they bound. There was also no difference in sperm acrosome status among the four groups. In summary, sorting reduced sperm binding to the complex matrix around oviductal cell aggregates but sperm binding to purified soluble oviduct glycans was not affected. The requirement for higher affinity and motility to bind glycans immobilized on oviduct cells may explain this difference. The reduction in sperm fertility observed following sex-sorting may be explained partially by a reduced or altered ability to bind to the oviduct epithelium.

Effect of sex-sorting and cryopreservation on the post-thaw sperm quality of Iberian red deer spermatozoa

This study investigated the effect of sex-sorting and cryopreservation on post-thaw characteristics and fertility of red deer (Cervus elaphus) sperm for the first time. Semen was collected by electroejaculation from 10 mature stags during the breeding season, and each ejaculate split into four experimental groups: Bulk sorted spermatozoa, sorted but not sexed (BSS); sorted high purity X-spermatozoa (XSS); sorted high purity Y-spermatozoa (YSS); and, control non-sorted spermatozoa (NS). Following, all samples were frozen over liquid nitrogen. Two straws per stag and sample type were analyzed immediately post-thaw and following a 2-h incubation period at 37 °C. Post-thaw total motility (TM) as assessed by CASA was not different (P < 0.05) among NS, BSS and YSS sperm. For XSS, post-thaw TM was lower (39%, P < 0.05) than that for NS (54%) or BSS (50%), but similar (P > 0.05) to that of YSS (47%) sperm. The percentage of apoptotic spermatozoa as assessed by PI/YO-PRO-1 and flow cytometry analysis, was higher (17%, P ≤ 0.05) for XSS sperm than NS (12%), BSS (13%) and YSS (14%) sperm. Following incubation there were no differences (P > 0.05) in TM or percent apoptosis among treatments. Post-thaw chromatin stability calculated as the DNA fragmentation index (%DFI) was similar among treatments; following incubation %DFI increased in all except YSS, which displayed the lowest value (P < 0.05). Artificial insemination of synchronized hinds yielded 44, 52 and 62% delivery rates for YSS, NS and standard frozen-thawed sperm, respectively (P < 0.05). Notably, 93 and 55% of fawns born were males for the YSS and NS spermatozoa, respectively (P < 0.05). In summary, Y-sorted sperm displayed acceptable post-thaw sperm evaluation parameters and the expected offspring sex ratio. More studies are needed to understand the source of sperm damage that may compromise the fertility of Y-sorted red deer sperm.

The Effect of Oxidative Stress on Thawed Bulk-Sorted Red Deer Sperm.

The aims of this study were to assess the effects of the sex-sorting process on post-thaw sperm quality as well as on induced oxidative stress damage (H2 O2 0mm=H000; H2 O2 50mm=H050; H2 O2 100mm=H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non-sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p<0.05) for NS (59%±3.3) than BSS (36.9%±5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p<0.05) for BSS (21.6%±5.0) than NS sperm (14.6%±1.2). The presence of H2 O2 increased DNA damage in NS (H000 =4.1%±0.9; H050=9.3%±0.7; and H100=10.9%±2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p<0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5±4.8 vs 21.1±3.9 and BSS: 33.3±8.1 vs 8.9±1.8). These results demonstrate that the sperm-sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non-sorted sperm. Moreover, addition of GSH at 1mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.

Sex-sorting of spermatozoa affects developmental competence of in vitro fertilized oocytes in a bull-dependent manner.

The aim of the present study was to clarify if flow-cytometric sex-sorting of bovine sperm affected in vitro blastocyst production in different bulls, either in terms of its ability to fertilize the oocyte or by interfering with post-fertilization embryo development. We performed in vitro fertilization (IVF) using both commercially available frozen-thawed X-sorted and non-sorted sperm of 4 Holstein bulls at 3 concentrations (1 × 106, 2 × 106, and 5 × 106 sperm/ml). When fertilization rates were compared, a variation in fertilization rates among different sperm concentrations was detected in 2 bulls, with similar results for X-sorted and non-sorted sperm. However, we found no evidence that the fertilization rates were affected by the sorting process. To investigate effects on embryo development, we determined the optimum sperm concentration for IVF in each bull, which resulted in similar fertilization rates among bulls. We next performed IVF using both X-sorted and non-sorted sperm of the 4 bulls at their optimum sperm concentration and compared in vitro embryo development. Cleavage rates with X-sorted sperm were similar to their non-sorted counterparts. However, significantly reduced blastocyst development was associated with the use of X-sorted sperm in one bull, whereas in the other three bulls, blastocyst development after IVF with X-sorted and non-sorted sperm was similar. In conclusion, in our system, X-sorting affects in vitro blastocyst production by reducing the developmental competence of fertilized oocytes rather than affecting the fertilization ability of the sperm. However, the occurrence of this phenomenon varies among bulls.

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.

237 THE EFFECTS OF BULLS AND X-SORTING OF SPERM ON THE ACCURACY OF NONINVASIVE CRITERIA TO PREDICT BLASTOCYST FORMATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

Previously, an early first cleavage and a second cleavage after IVF with a normal cleavage pattern defined by even blastomeres without fragments or protrusions was found to be a potent marker for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLoS ONE 7, e36627). The aim of this study was to investigate the effects of bulls and X-sorting of sperm on the ability of these simple noninvasive markers to predict the potency of bovine IVF embryos to develop to the blastocyst stage in vitro. Immature oocytes were matured in TCM199 supplemented with 0.02 armour unit mL–1 FSH and 5% calf serum at 38.5°C in 5% CO2 and 95% air for 22 to 23 h. After maturation, oocytes were inseminated with either of non-sorted frozen-thawed sperm from 3 bulls (A–C) or X-sorted sperm of bull A. Putative zygotes were cultured (IVC) in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually by time-lapse cinematography (CCM-1.3Z; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). First and second cleavage kinetics and pattern were categorized according to Sugimura et al. (2012). For each bull, blastocyst development from embryos possessing the following 3 selection markers was compared: (marker 1) the first cleavage within 28 h after IVF, (marker 2) marker 1 combined with 2 even blastomeres without fragments or protrusions, and (marker 3) marker 2 combined with the second cleavage within 50 h after IVF with ≥6 even blastomeres without fragments or protrusions, respectively. Data were analysed by the Yates' corrected chi-square test. A total of 823 oocytes were used in at least 3 replications. When non-sorted sperm was used for IVF, there was not difference (P &gt; 0.05) in total blastocyst formation rates on Day 8 (Day 0 = IVF) among bulls (ranging between 49.5 and 60.8%); however, blastocyst formation rate of embryos generated from X-sorted sperm of bull A (39.5%) was lower (P &lt; 0.05) compared with other groups despite of similar cleavage rates. Embryos having marker 3 criteria developed to the blastocysts stage at significantly higher rates than those having marker 1 criteria in case of non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A (75.9, 87.0, 90.0, and 75.0% v. 59.5, 62.2, 63.6, and 46.3%, respectively). In groups produced from non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A, blastocyst development rates of embryos with marker 2 criteria (73.7, 75.0, 90.0, and 65.8%, respectively) were higher (P &lt; 0.05) than those of embryos having marker 1 criteria but did not differ significantly from those with marker 3 criteria. Our results reveal that a first cleavage within 28 h after IVF to 2 even blastomeres without fragments or protrusions are potent predictive markers of the developmental competence of bovine embryos to the blastocyst stage regardless of bulls and sperm sorting.Research was partly supported by JSPS KAKENHI (26450388).

319 APPROACHES TO IMPROVE INTRACYTOPLASMIC SPERM INJECTION MEDIATED TRANSGENESIS AND MAXIMIZE THE USE OF SEX-SORTED SPERM IN BOVINE

Intracytoplasmic sperm injection (ICSI) mediated transgenesis is an effective tool for transgenic animal production. However, ICSI in cattle remains inefficient. In this work, we assayed approaches to improve egfp expressing blastocysts production by ICSI: the sperm pretreatment with heparin and l-glutathione (Hep-GSH), the use of sex-sorted sperm (SS), the refrozen/thawing of SS sperm, and the combination of these. Quality of ICSI blastocysts was analysed by studying the expression of 4 genes, and the rates of DNA fragmentation. Cumulus-oocyte complexes from slaughtered cow ovaries were in vitro-matured for 21 h. Nonsorted (NS) and sex-sorted (SS) frozen straws were thawed. Some of them were incubated with 80 μM Hep-15 mM GSH for 20 h (Hep-GSH+). The Hep-GSH-control group was not pretreated. Semen samples were co-incubated with 50 ng µL–1 of pCX-EGFP for 5 min before ICSI. Moreover, the SS sperm that are usually discarded after ICSI were cryopreserved and used for ICSI after a second thawing (ICSI SS refrozen). The ICSI NS, sham, and diploid parthenogenetic (Diplo PA) controls were included. Oocytes were activated with 5 µM ionomycin for 4 min, TCM-199 for 3 h (except for diploid PA), and 1.9 mM DMAP for 3 h. Cleavage and blastocyst/egfp expression rates were evaluated on Days 2 and 7 post-ICSI, respectively. Results are shown in Table 1. Relative expression of HMGN1, GLUT5, AQP3, and OCT4 genes from ICSI NS Hep-GSH+ and IVF blastocysts were compared by qPCR. Data were analysed by the pair-wise fixed reallocation randomisation test. None of the 4 genes showed differences between groups. The DNA fragmented nucleus index/blastocyst cell numbers were determined by TUNEL assay, not showing differences between groups (Kruskal–Wallis test, P ≤ 0.05). Means ± s.d. were 29 ± 17/91 ± 27 for ICSI Hep-GSH+; 27 ± 15/63 ± 34 for ICSI Hep-GSH–; 28 ± 17/68 ± 17 for ICSI SS, 28 ± 13/75 ± 24 for ICSI SS refrozen; and 21 ± 13/105 ± 59 for IVF SS control. The Hep-GSH pretreatment can increase blastocyst and transgene expressing blastocysts rates after TM-ICSI, except when SS semen is used. Interestingly, the use of SS sperm for ICSI can be maximized by cryopreservation and reuse of discarded sperm cells. The parameters analysed in this work indicate that the proposed approaches do not affect blastocyst quality. Therefore, Hep-GSH pretreatment of NS sperm and refrozen SS sperm could be applied for TM-ICSI in bovine for the production of transgenic animals. Table 1.In vitro development and egfp expression of ICSI embryos fertilized with nonsorted (NS) and sex-sorted (SS) sperm pretreated with Hep-GSH, refrozen, or both

Sperm sex-sorting and preservation for managing the sex ratio and genetic diversity of the southern white rhinoceros (Ceratotherium simum simum)

White rhinoceros ejaculates (n=9) collected by electroejaculation from four males were shipped (10°C, 12h) to develop procedures for the production of chilled and frozen-thawed sex-sorted spermatozoa of adequate quality for artificial insemination (AI). Of all electroejaculate fractions, 39.7% (31/78) exhibited high quality post-collection (≥70% total motility and membrane integrity) and of those, 54.8% (17/31) presented reduced in vitro quality after transport and were retrospectively determined to exhibit urine-contamination (≥21.0μg creatinine/ml). Of fractions analyzed for creatinine concentration, 69% (44/64) were classified as urine-contaminated. For high quality non-contaminated fractions, in vitro parameters (motility, velocity, membrane, acrosome and DNA integrity) of chilled non-sorted and sorted spermatozoa were well-maintained at 5°C up to 54h post-collection, whereby >70% of post-transport (non-sorted) or post-sort (sorted) values were retained. By 54h post-collection, some motility parameters were higher (P<0.05) for non-sorted spermatozoa (total motility, rapid velocity, average path velocity) whereas all remaining motion parameters as well as membrane, acrosome and DNA integrity were similar between sperm types. In comparison with a straw method, directional freezing resulted in enhanced (P<0.05) motility and velocity of non-sorted and sorted spermatozoa, with comparable overall post-thaw quality between sperm types. High purity enrichment of X-bearing (89±6%) or Y-bearing (86±3%) spermatozoa was achieved using moderate sorting rates (2540±498X-spermatozoa/s; 1800±557Y-spermatozoa/s). Collective in vitro characteristics of sorted-chilled or sorted-frozen-thawed spermatozoa derived from high quality electroejaculates indicate acceptable fertility potential for use in AI.

138 COMPARISON OF KINETICS AND PATTERNS OF THE FIRST CLEAVAGE OF IN VIVO AND IN VITRO-MATURED HOLSTEIN OOCYTES AFTER IN VITRO FERTILIZATION WITH X-SORTED SPERM

Previously, it was reported that a high rate of good quality blastocysts were produced by IVF of in vivo-matured oocytes, obtained by ovum pick up (OPU) after superstimulation in Holstein cows, using X-sorted sperm (Matoba et al. 2012 Reprod. Domest. Anim. 47, 515). In this system, an early first cleavage within 28 h after IVF was found to be a potent marker for the selection of embryos with high developmental competence (Matoba et al. 2013 Reprod. Fertil. Dev. 25, 266). However, we have limited knowledge on the timing and normality of embryonic cleavages in in vitro-matured oocytes after IVF. The purpose of the present study was to compare the kinetics and patterns of the first cleavage of in vivo- and in vitro-matured bovine oocytes after IVF with X-sorted sperm. In vivo-matured oocytes (Group A) were collected by OPU from non-lactating Holstein cows just before ovulation after superstimulation. Immature oocytes were either collected by OPU without hormonal treatment or by aspiration of ovaries at the local abattoir and matured in vitro (Group B or C). All the oocytes were inseminated with 5 × 106 sperm mL–1 of X-sorted sperm, except half of oocytes in Group C inseminated by non-sorted sperm (Group D) and cultured in CR1aa supplemented with 5% calf serum and 0.25 mg mL–1 of linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually using a microwell culture dish and time-lapse cinematography (Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken in every 15 min during the IVC period and analysed by time-lapse cinematography software. Cleavage pattern was categorized as normal (2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with fragment or protrusion and those dividing into 3–5 blastomeres) at the first cleavage. Data were analysed by ANOVA, chi-squared, or discriminant function. A total of 268 cleaved embryos were used. The blastocyst rate in Group A was higher than in Groups B and C (61.3 v. 40.0 and 25.0%, respectively; P &lt; 0.05). The timing of first cleavage was longer in Group A compared with Groups C and D (28.3 ± 3.8 v. 27.6 ± 3.8 and 26.7 ± 1.9 h, respectively) and in Group B (28.1 ± 4.0 h) compared with in Group D (P &lt; 0.05). Higher rates of normal cleavage were observed in Groups A, B, and D than in Group C (53.5, 44.4, and 54.8 v. 16.7%, respectively; P &lt; 0.01). The frequency of blastocysts derived from the early (28.3 h) and normal pattern cleaving oocytes were greater in Groups A and B than in Group C (29.0 and 20.0 v. 8.3%, respectively; P &lt; 0.05) and similar in Group D (22.6%). Our results reveal that IVF embryos produced from in vivo-matured oocytes with sex-sorted sperm had superior normality than those produced from in vitro-matured oocytes and similar normality to embryos inseminated with non-sorted sperm. Supported by the Research and Development projects for application in promoting new policy of agriculture, forestry and fisheries (22016) and by JSPS and HAS under the Japan-Hungary Research Cooperative Program.

Birth of Kids After Artificial Insemination with Sex‐Sorted, Frozen‐Thawed Goat Spermatozoa

Successful sex-sorting of goat spermatozoa and subsequent birth of pre-sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm-sorting (using a modified flow cytometer, MoFlo SX(®) ) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n=2 males) were (i) collected into Salamon's or Tris catch media post-sorting and (ii) frozen in Tris-citrate-glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25ml pellets on dry ice or 0.25ml straws in a controlled-rate freezer. Post-sort and post-thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC-PNA). Sex-sorted goat spermatozoa frozen in pellets displayed significantly higher post-thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex-sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex-sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p>0.05) to non-sorted goat spermatozoa, non-sorted ram spermatozoa and sex-sorted ram spermatozoa. Following intrauterine artificial insemination with sex-sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non-sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3:1 (F:M). This study demonstrates for the first time that goat spermatozoa can be sex-sorted by flow cytometry, successfully frozen and used to produce pre-sexed kids.

Dimethyl formamide improves the postthaw characteristics of sex-sorted and nonsorted stallion sperm

Cryopreserved, sex-sorted stallion sperm has been shown to have poor fertility. During this study, the effects of cryoprotectant (glycerol [GLY] and dimethyl formamide [DMF]), cryoprotectant equilibration time (0, 30, 60, 90, or 120 minutes), and cryoprotectant concentration (2%, 3%, or 4% vol/vol) on stored sex-sorted and stored nonsorted stallion sperm were evaluated. Total motility, viability, and DNA integrity (determined using sperm chromatin structure assay) of sperm were assessed after thawing. Equilibration for 90 minutes improved total motility (33.8%) compared with 0 (28.5%) or 120 minutes (29.8%; P < 0.05), though viability was higher after 120 minutes (33.1%) compared with 0 (30.5%) or 30 minutes (31.0%; P < 0.01). The viability of nonsorted sperm decreased as cryoprotectant concentration increased (P < 0.001), and total motility of nonsorted sperm was higher when DMF alone was used (15.8%, 16.6%, and 24.0% for GLY, GLY and DMF, and DMF respectively; P < 0.001). Sex sorting was detrimental to the postthaw quality of sperm; at 45 minutes after thawing, total motility of nonsorted sperm was higher than that of sex-sorted sperm (37.4% vs. 5.6%; P < 0.001), the viability of sex-sorted sperm was lower than that of nonsorted sperm (12.4% vs. 30.0%; P < 0.001, averaged over postthaw time), and sex-sorted sperm had higher detectable DNA fragmentation index (DFI) (63.6% vs. 11.3%, P < 0.001) and mean DFI (285.1 vs. 211.3, P < 0.001) than nonsorted sperm. The viability of sex-sorted sperm was improved by GLY and DMF or DMF compared with GLY (22.6%, 25.3%, and 19.3%, respectively; P < 0.05), and the DNA integrity of sex-sorted sperm was improved by the use of DMF compared with GLY (detectable DFI, 60.2 vs. 66.8, P < 0.05; and mean DFI, 280.9 vs. 289.2, P < 0.05, respectively). In conclusion, postthaw characteristics of stored sex-sorted and stored nonsorted stallion sperm were improved by the use of DMF as a cryoprotectant, though the parameters to benefit differed between sorted and nonsorted sperm.

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.

Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

The Effect of Glycerol Concentrations on the Post‐thaw In Vitro Characteristics of Cryopreserved Sex‐sorted Boar Spermatozoa

The objective of this study was to optimize protocols for the cryopreservation of sex-sorted boar spermatozoa. In the experiment 1, we evaluated the effects of a standard boar sperm cryopreservation procedure (3% final glycerol concentration) on the in vitro characteristics of sex-sorted sperm frozen at low sperm concentrations (20 × 10(6) sperm/ml; S20 group). Non-sorted spermatozoa frozen at 1000 × 10(6) (C1000 group) and 20 × 10(6) (C20 group) sperm/ml were used as the freezing control groups. In experiment 2, the effects of different final glycerol concentrations (0.16%, 0.5%, 1.0%, 2.0% and 3.0%) on post-thaw quality of the S20 and C20 groups were evaluated. In both experiments, the samples were evaluated prior to freezing (5°C) and at 30, 90 and 150 min after thawing. Experiment 1 indicated that freezing sperm at low concentrations decreased (p < 0.05) the total motility (TM) and progressive motility (PM) at 90 and 150 min after thawing regardless of whether the sperm were sorted or not. However, the sperm membrane integrity was not affected at any evaluation step. Inexperiment 2, significant effects on the TM and PM because of increased glycerol concentrations in the S20 and C20 groups were observed only at 90 and 150 min after thawing. The samples frozen in 3% glycerol showed lower (p < 0.05) TM and PM values when compared to those frozen in the presence of 0.5% and 1% glycerol. In both experiments, non-sorted control samples displayed higher percentages of spermatozoa with damaged DNA than sorted spermatozoa. In conclusion, the optimization of cryopreservation conditions by decreasing the glycerol concentrations can improve post-thaw motility of sex-sorted spermatozoa frozen at low concentrations.

Timing of insemination using sex-sorted sperm in embryo production with Bos indicus and Bos taurus superovulated donors

Two experiments were designed to evaluate the effect of different insemination times (12 and 24 h or 18 and 30 h) and different types of semen (sex-sorted or non-sorted sperm) on embryo production in Nelore ( Bos indicus) and Holstein ( Bos taurus) superstimulated donors. In the first experiment, hormonal superstimulation of ovarian follicular development in Nelore donors ( n = 71) was performed in randomly allocated animals to one of the three treatment groups, and they were inseminated at 12 and 24 h after an ovulatory stimulus with pLH treatment was applied, either with sex-sorted (4.2 × 10 6 sperm/insemination; S12/24; n = 17) or non-sorted sperm (20 × 10 6 sperm/insemination; NS12/24; n = 18), or they were inseminated at 18 and 30 h using sex-sorted sperm (4.2 × 10 6 sperm/insemination; S18/30; n = 19). A greater number of transferable embryos were found when sex-sorted sperm was used to inseminate the animals at 18 and 30 h (4.5 ± 3.0) compared to insemination at 12 and 24 h (2.4 ± 1.8; P < 0.001). However, a greater embryo production (6.8 ± 2.6) was obtained with non-sorted sperm. In the second experiment, the same insemination times and semen types were used in lactating high-production Holstein cows ( n = 12). A crossover design was employed in this trial. A lesser embryo production ( P = 0.007) was found in Holstein donors that were inseminated using sex-sorted sperm at 12 and 24 h (4.6 ± 3.0) compared to non-sorted sperm (8.7 ± 2.8). However, intermediate results were obtained when the inseminations with sex-sorted sperm were performed at 18 and 30 h (6.4 ± 3.1). These results supported the current hypothesis that it is possible to improve embryo production using sex-sorted sperm in B. indicus and B. taurus superstimulated donors when the inseminations are performed near the same time as time-synchronized ovulations. However, the embryo production for timed artificial insemination (TAI) with sex-sorted sperm was still less than the production with non-sorted sperm.