Sex-sorted Ram Spermatozoa Research Articles

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.

Seminal plasma proteins do not consistently improve fertility after cervical insemination of ewes with non-sorted or sex-sorted frozen - thawed ram spermatozoa

The effect of supplementation of sex-sorted and non-sorted spermatozoa with seminal plasma protein (SPP) on fertility after cervical insemination was examined in the present study. Spermatozoa were sorted into high purity X and Y chromosome-bearing spermatozoa or not sorted and then either supplemented with SPP (>10 kDa) before freezing and/or after thawing (non-sorted only) or processed without supplementation. Inseminations were performed over 2 days with ewes receiving 100 or 25 million motile non-sorted spermatozoa in the cervix or uterus, respectively, or two cervical inseminations of 3.5 million motile sorted spermatozoa. Pregnancy rates in cervically inseminated ewes were unaffected by supplementation of sorted or non-sorted spermatozoa with SPP before freezing compared with no supplementation. The effect of post-thaw supplementation of non-sorted spermatozoa with SPP on pregnancy rates after cervical insemination varied with the day of insemination (P < 0.05); fertility was similar to laparoscopic insemination on Day 1 (56.0 +/- 10.2% v. 58.6 +/- 10.1%), but not on Day 2 (23.1 +/- 7.4% v. 66.7 +/- 9.2%). In conclusion, under the conditions of the present study, SPP did not consistently improve pregnancy rates after cervical insemination with frozen-thawed ram spermatozoa. This is the first report of pregnancies (5/56 ewes inseminated) after cervical insemination with frozen-thawed sex-sorted ram spermatozoa. Although the success rate is low, the findings are encouraging because ewes inseminated with the sex-sorted spermatozoa received only 7% of the recommended dose (100 million motile) for cervical insemination of frozen-thawed spermatozoa.

364 QUANTIFICATION OF DEVELOPMENTALLY IMPORTANT GENE TRANSCRIPTS EXPRESSED IN EMBRYOS PRODUCED IN VIVO AND IN VITRO USING SEX-SORTED AND NON-SORTED RAM SPERMATOZOA

The AI of sex-sorted spermatozoa results in decreased levels of fertility in most species. This is not the case in sheep, where low-doseAI of sex-sorted ram spermatozoa produces similar, if not superior levels of fertility to non-sorted controls (Beilby et al. 2009 Theriogenology, 71, 829-835). In an effort to provide insight into the molecular basis for this difference in fertility between species, the aim of the present study was to examine the impact of sex-sorting technology on ovine embryo gene expression. After semen collection, ejaculates (n = 8) were split and either sex-sorted by flow cytometry and frozen, or diluted and frozen (non-sorted control). Embryos were produced in vivo by inseminating superovulated ewes with either X- or Y-chromosome enriched spermatozoa, or non-sorted control spermatozoa, and collected by flushing uterine horns on Day 6 after AI. Embryos were produced in vitro by using established oocyte in vitro maturation and in vitro fertilization procedures (using X, Y or non-sorted spermatozoa), and cultured in vitro for 6 days. The relative abundance of Glut-3, G6PD, SUV39H1, DnMT3a, and HSP70 was measured in high grade blastocysts (in vivo Day 6: n = 23; in vitro Day 6: n = 21) using quantitative, real-time PCR (iCycler5TM, BioRad, Hercules, CA, USA). Blastocyst cell numbers were quantified to ensure embryos were at a similar stage of development. The sex of all embryos was identified by PCR to allow comparison between treatments. Fold differences in gene expression, acquired through the A ACt method, were calculated and compared by an ANOVA. The expression of HSP70 was up-regulated in in vitro embryos derived from sex-sorted spermatozoa compared to those produced in vivo (P &lt; 0.05). For all other genes examined, there was no effect of sex or sperm treatment on gene expression. Glut-3, G6PD, SUV39H1, andDnMT3a were all up-regulated in in vitro embryos compared with in vivo embryos (P &lt; 0.05). These results suggest that fertilization with sex-sorted ram spermatozoa does not result in aberrant patterns of gene expression in embryos produced in vivo. The data from the present study provide further evidence that in vitro culture induces epigenetic modification within the embryonic genome, when compared to the in vivo physiological standard. It would be of interest to conduct a similar in vivo study in cattle, where sex-sorting technology has not been as biologically successful. The altered expression of HSP70, which is associated with cellular stress, may demonstrate a cumulative impact of in vitro reproductive technologies on the preimplantation embryo. Research supported by XY, Inc.

Sperm sexing in sheep and cattle: The exception and the rule

Flow cytometric sorting for the preselection of sex has progressed considerably in the 20 years since its inception. This technique has allowed the production of pre-sexed offspring in a multitude of species and become a commercial success in cattle around the world. However, due to the stress inherent to the sex-sorting process, sex-sorted spermatozoa are widely recognized as functionally compromised in terms of their fertilizing lifespan within the female reproductive tract as a result of reduced motility and viability and changed functional state. These characteristics, when compared to non-sorted controls, are manifest in vivo as lower fertility. However, improvements to the technology and a greater understanding of its biological impact have facilitated recent developments in sheep, showing sex-sorting is capable of selecting a functionally superior population in terms of both in vitro and in vivo function. These results are reviewed in the context of recent developments in other species and the reasons for success after artificial insemination with sex-sorted ram spermatozoa are discussed.

Application of seminal plasma in sex-sorting and sperm cryopreservation

Substantial dilution of boar semen during processing decreased the concentration of seminal plasma, perhaps contributing to the decline in sperm quality after cryopreservation and sex-sorting. Results of replacing seminal plasma in investigations from many laboratories have been contradictory. Results and discussion here suggest that whereas membrane status can be influenced by seminal plasma, the action of its various components, both positive and negative, is determined in part by the membrane status of the spermatozoa to which it is being exposed. Although progress has been made in identifying components of seminal plasma responsible for its protective effect (notably PSP-I/II spermadhesin for sex-sorted boar spermatozoa), little is known (in any species) regarding how external factors may influence their levels, and their functionality, in seminal plasma. It is noteworthy that seminal plasma is beneficial to post-thaw quality of sex-sorted ram spermatozoa only when added before freezing, not after thawing. Therefore, the action of seminal plasma and its components is dependent on sperm-related factors, in particular the type of processing to which they have been previously exposed. Further research is needed to unravel these biological complexities, and then characterise and synthesise useful proteins within seminal plasma.

Influence of supplementing diet with Oleic and Linoleic acid on the freezing ability and sex-sorting parameters of ram semen

Abstract In an effort to improve the cryosurvival of both non-sorted and sex-sorted ram spermatozoa the effect of supplementing the ram diet with Oleic and Linoleic acid, in the form of extra virgin olive oil and sunflower oil, respectively, was assessed. Rams ( n = 4/group) were fed either (i) a standard maintenance diet (Control), (ii) maintenance diet + 5% (w/w) sunflower oil (Linoleic), or (iii) maintenance diet + 5% (w/w) extra virgin olive oil (Oleic) for a period of 6 weeks. The effect of these diets on the post-thaw (incubated 37 °C, 6 h) motility characteristics (as measured by CASA) of non-sorted, frozen-thawed ram spermatozoa were assessed every 2 weeks. The sex-sorted, frozen–thawed spermatozoa were assessed at the end of the 6 week trial period in the same manner. Linoleic and Oleic diets had a negative impact ( P P P

Embryo production from superovulated sheep inseminated with sex-sorted ram spermatozoa

An experiment was undertaken to assess the fertilizing capacity of sex-sorted, frozen-thawed ram spermatozoa, artificially inseminated into superovulated ewes, and the quality and survivability of the resultant pre-sexed embryos. Synchronized (intravaginal progestagen pessary and GnRH) donors were superovulated using PMSG and repeat ovarian stimulation with FSH before insemination. Ewes ( n = 67) were inseminated with either 30 × 10 6 or 15 × 10 6 motile non-sorted (control) or 15 × 10 6 motile sex-sorted (sorted) frozen-thawed spermatozoa (control: C 30 or C 15; sorted: S 15, respectively) and the resultant embryos transferred immediately into synchronized recipients ( n = 160). The percentage of transferable embryos, pregnancy rate and embryo survival were similar ( P > 0.05) across all treatments. Oocyte cleavage rate was higher for ewes inseminated with S 15 (172/230; 74.8%; P < 0.05) than for C 15 (97/151; 64.2%) or C 30 (89/141; 63.1%) spermatozoa. Of the lambs resulting from embryos produced with sex-sorted spermatozoa, 86/93 (92.5%) were born of the predicted sex. This study demonstrated for the first time that pre-sexed offspring derived from superovulated sheep can be produced following transfer of embryos. Furthermore, sex-sorting by flow cytometry did not compromise the in vivo fertilizing capacity of ram spermatozoa in superovulated sheep, nor did it affect the quality or survivability of the resultant embryos.

The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa

In an effort to improve the number of functional spermatozoa following sex-sorting and cryopreservation, the effects on in vitro sperm characteristics of the additives: (i) catalase (pre-sorting); (ii) cholesterol-loaded cyclodextrins (CLCs; pre-sorting); and (iii) seminal plasma (post-thawing) were investigated. For all experiments, spermatozoa (three males, n = 3 ejaculates/male) were processed using a high speed flow cytometer before cryopreservation, thawing and incubation for 6 h. Catalase had no effect ( P > 0.05) on post-thaw motility characteristics (as measured by CASA) of sex-sorted ram spermatozoa, but pre-sort addition of CLCs reduced ( P < 0.05) sperm quality after post-thaw incubation for 0 h (motility), 3 h (motility, average path velocity, viability and acrosome integrity) and 6 h (motility, average path and curvilinear velocity, straightness, linearity, viability and acrosome integrity). Seminal plasma had a differential effect ( P < 0.001) on sex-sorted and non-sorted spermatozoa. Post-thaw supplementation of increasing levels of seminal plasma caused all motility characteristics of sex-sorted, frozen-thawed spermatozoa to decline ( P < 0.05); conversely, non-sorted, frozen-thawed spermatozoa exhibited improvements ( P < 0.05) in motility, viability, acrosome integrity and mitochondrial respiration. In summary, incorporation of catalase, CLCs and seminal plasma into the sorting protocol failed to improve post-thaw sperm quality and, consequently efficiency of sex-sorting of ram spermatozoa. The paradoxical effect of seminal plasma supplementation on the in vitro characteristics of ram spermatozoa provides further evidence that sex-sorting by flow cytometry produces a selected population of cells with different functions compared with non-sorted spermatozoa.

The effect of gamete co-incubation time during in vitro fertilization with frozen-thawed unsorted and sex-sorted ram spermatozoa on the development of in vitro matured adult and prepubertal ewe oocytes

In vitro matured adult (Experiment 1) and prepubertal (Experiment 2) ewe oocytes were co-incubated with unsorted or sex-sorted frozen-thawed spermatozoa for 2 to 3 h (short) or 18 to 20 h (long) to determine the effects of reducing the gamete co-incubation time during IVF on subsequent embryonic development in vitro. For oocytes derived from adult ewes, there were no differences in oocyte fertilization and cleavage at 24 h post insemination (hpi) between types of spermatozoa or co-incubation times ( P > 0.05). By 48 hpi, oocyte cleavage was higher after a short (390/602, 64.8%) compared with a long (381/617, 61.7%) co-incubation ( P < 0.05), and was not significantly different for unsorted (266/372, 71.5%) and sex-sorted (505/849, 59.9%) spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (150/266, 56.4%) and sex-sorted (295/505, 58.4%) spermatozoa, but was higher after a short (240/390, 61.5%) than long (205/381, 53.8%) co-incubation ( P < 0.05). Oocyte development to the blastocyst stage was not different for unsorted (150/372; 40.3%) and sex-sorted (295/847; 34.8%) spermatozoa but was significantly increased by a short (240/602, 39.9%) compared with a long (205/617, 33.2%) co-incubation. Fertilization of oocytes from prepubertal ewes was similar for types of spermatozoa and for duration of co-incubation. Oocyte cleavage (48 hpi) was similar for a short (241/377, 63.9%) and long (226/349, 64.8%) co-incubation with unsorted spermatozoa, but was increased ( P < 0.05) by a long co-incubation (286/500, 57.2% versus 163/517, 31.5%) with sex-sorted spermatozoa. Blastocyst formation from cleaved oocytes was similar for unsorted (230/467, 49.3%) and sex-sorted (186/449, 41.4%) spermatozoa, and a short (200/404, 49.5%) or long (216/512, 42.1%) co-incubation. However, oocyte development to the blastocyst stage was higher ( P < 0.05) after IVF with unsorted (230/726, 37.1%) than sex-sorted (186/1017, 18.3%) spermatozoa. Reducing the duration of gamete co-incubation did not deleteriously affect the in vitro development of adult and prepubertal ewe derived oocytes after IVF with unsorted and sex-sorted spermatozoa. In general, sex-sorting had no substantial influence on fertilization and embryo development rates.

330THE EFFECT OF SHEATH FLUID ON THE QUALITY OF SEX-SORTED RAM SPERMATOZOA

The fertility of sexed frozen-thawed ram spermatozoa at low AI doses remains below commercially acceptable levels (Maxwell WMC et al. 2003 Theriogenology 59, 511 abst). The aim of the present study was to determine if the use of an artificial seminal plasma sheath fluid could attenuate the stress of dilution during flow cytometry and thus improve the functional capacity of sexed ram spermatozoa. Semen was collected from 3 Merino rams, processed for sex-sorting (Hollinshead FK et al. 2002 Reprod. Fertil. Dev. 14, 503–508), and then allocated to 2 treatment groups according to the sheath fluid used within the flow cytometer: (i) a tris-citrate-fructose diluent (TRIS), or (ii) an artificial seminal plasma diluent (ASP) (ODonnell JM 1969 J. Reprod. Fert. 19, 207–209). Spermatozoa were sorted using a modified high speed cell sorter (SX MoFlo®, DakoCytomation, Fort Collins, USA) without separation of X and Y bearing gametes, and then frozen and thawed using established protocols (Hollinshead FK et al. 2002). Motility characteristics (HTM-IVOS; Hamilton-Thorne, Beverly, USA) and acrosome integrity (FITC-PNA) were assessed throughout. Statistical analyses were conducted by ANOVA. Results indicate that in all but one instance (VSL pre-freeze), TRIS spermatozoa exhibited higher (P&amp;lt;0.05) total motility (TM), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) compared to ASP spermatozoa (Table 1). Conversely, ASP treatments displayed enhanced straightness (STR) and linearity (LIN) pre-freezing and immediately post-thaw. In addition, the percentage of hyperactivated (HA) spermatozoa prior to freezing (± SEM) was lower (P&amp;lt;0.05) for ASP (3±1.1) than for TRIS (20±3.4), although post-thaw the differences were NS. Acrosome integrity remained similar (P&amp;gt;0.05) among treatments before and after thawing (range: 92.3–95.9% intact). In conclusion, the ASP sheath fluid resulted in decreased TM, velocity and ALH of sex-sorted frozen-thawed ram spermatozoa, a clear indication of the superiority of TRIS sheath fluid. The partial improvement in STR, LIN and HA that the ASP affords sex-sorted ram spermatozoa does not offset the aforementioned negatives, suggesting ASP is unsatisfactory for use as a sheath fluid during sperm sorting. Research supported by XY, Inc. Table 1 The effect of sheath fluid on motility characteristics (±SEM)