Transvaginal aspiration, oocyte maturation and production of blastocysts and a pregnancy via ICSI in an endangered breed: Baudet du Poitou donkey.

Two Poitou donkey jennies were presented for clinical oocyte recovery and embryo production via intracytoplasmic sperm injection (ICSI). Both jennies underwent transvaginal ultrasound-guided follicle aspiration on two occasions. Recovered oocytes were held overnight then placed into maturation culture, using standard methods for mare oocytes. On the first replicate for both jennies, the oocytes were divided into two groups; one group was denuded and examined at 30 h culture (standard culture duration for mare oocytes) and the second was denuded and examined at 36 h culture. No oocytes with polar bodies were observed at either time. The oocytes were maintained in maturation culture until 46 h, at which time oocytes with polar bodies were observed. Semen was then prepared; oocytes underwent ICSI approximately 48 h after being placed into maturation culture. On the second replicate for both jennies, oocytes were cultured for maturation for 42 h, then denuded and subjected to ICSI at 46 h. Sperm preparation, injection and embryo culture were performed as for mare oocytes. Blastocyst rates per injected oocyte were 8/19 (42 %) overall, being 4/12 and 4/7 for the first and second TVAs, respectively. Blastocysts were vitrified. Three blastocysts were warmed and transferred to Poitou donkey jenny recipients. One embryonic vesicle was visualized on ultrasonography on embryo Day 12, which increased in size on Day 13 but was not present when examined on Day 14. These results demonstrate that oocyte recovery and ICSI are efficient for production of Poitou donkey blastocysts. To the best of our knowledge, this is the first report of production of blastocysts via ICSI in the Poitou donkey, and the first report of transfer of ICSI-produced embryos in the donkey. Further work is needed on factors affecting pregnancy after embryo transfer in the donkey.

Equine in vitro produced blastocysts: relationship of embryo morphology, stage and speed of development to foaling rate.

CONTEXT: Information on factors associated with developmental competence of equine in vitro -produced (IVP) blastocysts is lacking. AIMS: To determine the relationships of stage, grade, day of development, and specific morphological parameters of equine IVP blastocysts, to pregnancy and foaling rates. METHODS: Photomicrographs of 316 IVP embryos with known pregnancy outcomes were scrutinised individually by four observers. Inter-observer variation was assessed, and pregnancy outcome evaluated in relation to day of blastocyst development and assigned grade and stage. Individual component analysis was performed to determine the association of specific morphological parameters with foaling rate. KEY RESULTS: Overall pregnancy rate was 76.9% and foaling rate was 56.3%. The day of embryo development did not affect pregnancy rate but significantly affected foaling rate. Embryo stage did not affect foaling rate. Embryo grade affected foaling rate only for Day-9 embryos. Some morphological features in the bovine grading system did not predict outcome in equine IVP embryos. Significant individual parameters differed between Stage 5 and Stage 6 equine blastocysts. CONCLUSIONS: Day of blastocyst development is the major factor related to foaling rate for equine IVP embryos. Notably, there was no effect of embryo stage on foaling rate and no evidence that prolonging culture until embryos advance in stage increases foaling rate. The standard bovine grading system is not directly applicable to equine IVP embryos; equine-specific staging and grading systems are proposed. IMPLICATIONS: This information will allow laboratories to identify embryos with the highest developmental competence. Use of the proposed systems will increase consistency in embryo assessment among laboratories.

Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation†.

Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Transfer of three in vitro fertilization-produced blastocysts to recipient mares resulted in birth of three normal foals. When sperm were pre-incubated for 22 h, 47-79% of oocytes were fertilized after 1 h of co-incubation. Sperm pre-incubated for 15 min or 6 h before co-incubation yielded no fertilization at 1 h, suggesting that capacitation in this system requires between 6 and 22 h. Sperm assessed after 15 min, 6 h, or 22 h pre-incubation showed increasing protein tyrosine phosphorylation of the midpiece, equatorial band, and apical head; this pattern differed from that induced by high pH conditions and may denote functional equine sperm capacitation. Use of the final devised system, i.e., extended semen, with 22 h of sperm pre-incubation and 3 h of COC co-incubation, yielded 90% fertilization with a blastocyst rate of 74%. This is the first report of efficient and repeatable standard in vitro fertilization in the horse and the first report of in vitro production of blastocysts and resulting foals after in vitro fertilization.

Effect of day of estrus cycle at time of transvaginal follicle aspiration for oocyte recovery on rates of in vitro maturation and blastocyst production after intracytoplasmic sperm injection.

OBJECTIVE: To determine the effect of stage of estrus cycle (day after ovulation) at the time of transvaginal ultrasound-guided follicle aspiration (TVA) on parameters related to the success of in vitro equine embryo production. ANIMALS: 14 healthy mares were used; 11 completed the study and were included for analysis. PROCEDURES: Mares underwent TVA of all follicles ≥ 5 mm diameter at each of 3 timepoints: 7 days after ovulation, 14 days after ovulation, and S-DSF (subordinate to a dominant stimulated follicle), during estrus at 24 hours after gonadotropin administration. The 3 treatments were assigned to each mare in random order; mares underwent follicle growth and ovulation between treatments. Recovered oocytes were matured in vitro, subjected to intracytoplasmic sperm injection (ICSI), and cultured to the blastocyst stage in vitro. RESULTS: Total follicle numbers differed significantly between individual mares but did not differ between treatments. The number of follicles of different sizes significantly (P < 0.05) differed between treatments, with mares in the Day 7 treatment having more follicles 5 to 9 mm in diameter and fewer follicles 20 to 24 mm in diameter than mares in the other 2 treatments. After in vitro maturation culture, there were significantly more mature oocytes in the S-DSF treatment than in the other 2 treatments. There were no differences in blastocyst rate after ICSI among treatment groups. CLINICAL RELEVANCE: Timing of TVA for aspiration of S-DSFs may increase the number of mature oocytes available for ICSI. Understanding of the effects of timing of TVA will help veterinarians to maximize the efficiency of this procedure.

Culture protocols for horse embryos after ICSI: Effect of myo-inositol and time of media change.

In vitro production of horse embryos via intracytoplasmic sperm injection (ICSI) is a useful clinical and research technique. Current rates of blastocyst production are typically sub-optimal, and few methods to increase the rate of equine blastocyst development have been reported. Factors that might improve blastocyst production in a horse embryo culture system were explored. Myo-inositol is found in the horse oviduct and improves blastocyst development in other species, thus Experiment 1 was conducted to assess the effect of 10 mM myo-inositol added to Day 0-5 embryo culture medium, using horse oocytes recovered by transvaginal aspiration. Experiment 2 was conducted to investigate effects of exclusion of a standard post-ICSI holding step (culture for 30-60 min in M199-based medium). Experiment 3 was conducted using oocytes recovered from abattoir-derived ovaries, to evaluate effects of earlier transition (Day 4 vs. Day 5) to the second-step medium and of media refreshment at different time points (Day 3 and/or Day 7) during embryo culture. In Experiments 1 and 2, there were no differences (P > 0.05) between groups in blastocyst development (Exp. 1, 36.7 % and 39.2 %; Exp. 2, 41.5 % and 44.6 %). In Experiment 3, blastocyst development was not different (P > 0.05) for embryos refreshed at both Day 3 and 7 (10.8 %) or only at Day 7 (26.6 %), or those transferred to second-step medium on Day 4 or Day 5 (20.6 % and 18.5 %). Knowledge of culture procedures compatible with blastocyst formation in vitro is valuable to laboratories starting to develop procedures for ICSI in horses.

Factors affecting intracellular calcium influx in response to calcium ionophore A23187 in equine sperm.

BACKGROUND: Exposure to the calcium ionophore A23187 may present a "universal" sperm treatment for IVF, as it bypasses capacitation pathways. However, success in utilizing A23187 is variable, especially in equine spermatozoa. Notably, albumin is used during A23187 treatment but paradoxically is thought to suppress A23187 action. Essentially no critical data are available on the effects of A23187 and albumin concentrations, ratios, or addition protocols on changes in intracellular calcium ([Ca]i ) in any cell type. OBJECTIVE: To determine factors that affect the action of A23187 on [Ca]i in equine and murine spermatozoa. METHODS: Spermatozoa were loaded with Fluo-4 and changes in fluorescence after A23187 treatment were measured under various conditions using a microplate reader. RESULTS: Concentrations of bovine serum albumin (BSA) and A23187, type of BSA, makeup of A23187 stock solutions (i.e., 1° stock (DMSO) or 2° stock made with medium, water or DMSO), order of addition of spermatozoa and A23187, incubation of media before sperm addition, species of spermatozoa, and time of addition of BSA all affected [Ca]i in response to A23187 treatment. In equine spermatozoa already exposed to 10 µM A23187, addition of BSA to 33 mg/ml to "quench" the A23187 did not affect [Ca]i . When this concentration of BSA was added to spermatozoa exposed to 1 µM A23187, [Ca]i in murine spermatozoa returned to baseline, however, equine spermatozoa continued to exhibit increased [Ca]i . Addition of BSA to 33 mg/ml to media containing 1 µM A23187, prior to addition of spermatozoa, completely inhibited change in [Ca]i in both murine and equine spermatozoa. CONCLUSION: These results represent some of the first critical data on the effects of albumin and other procedural factors on A23187-induced changes in [Ca]i in any cell type. Our findings help to explain the variability in reported response of spermatozoa to A23187 among species and among laboratories.

Flow-cytometric analysis of membrane integrity of stallion sperm in the face of agglutination: the "zombie sperm" dilemma.

PURPOSE: To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect. METHODS: Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 µm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions. RESULTS: Using PI-PSA, measured membrane integrity (MI; live sperm) was dramatically lower than was total motility (TMOT), indicating spurious results ("zombie sperm"). Sperm aggregates were largely of motile sperm. Loss of motility after A23187 treatment was associated with disaggregation and increased MI. On disaggregation using LD-PSA, MI rose, and MI then corresponded with TMOT. In equine sperm incubated after A23187 treatment, as the percentage of live acrosome-reacted sperm increased, TMOT decreased to near 0. CONCLUSION: Flow cytometry assesses only individualized sperm; thus, agglutination of viable sperm alters recorded membrane integrity. As viable sperm become immotile, they individualize; therefore, factors that decrease motility, such as A23187, result in increased measured MI. Disaggregation before assessment allows more accurate determination of sperm membrane status; in this case we documented a mismatch between motility and live acrosome-reacted equine sperm that may relate to the poor repeatability of A23187 treatment for equine IVF. These findings are of profound value to future studies on sperm capacitation.

Histological characteristics of the gonads of pig fetuses and their relationship with fetal anatomical measurements.

The objective was to evaluate the histomorphometric characteristics of the testis and ovaries of pig fetuses at different gestational ages, as well as their correlation with some fetus measurements. Forty-four fetuses were separated for gender (male and female) and gestational age (50, 80 and 106days of gestation). After slaughter, fetuses had their body length, head and thoracic perimeters measured and their gonads submitted to histomorphometric analyses. The gonadal characteristics at different gestational ages were statistically compared, correlations with the fetal measurements were performed and equations to predict the gonadal characteristics from the fetal measurements were obtained. The testis weight logarithmically increased along pregnancy, whereas ovary weight increased in a linear manner. The cordonal length and number of Sertoli cells were positively correlated with the fetal measurements, being higher at 106days gestation, while the nuclear volume of these cells were negatively correlated. The total number of follicles was higher at day 80 and 106 of pregnancy. The number of oogonia decreased along the pregnancy, however, their nucleus size was increased. The number of follicles and volume of oogonia were positively correlated with the fetal measurements, while the number of oogonia was negatively correlated. Equations were obtained for the prediction of gonadal characteristics of fetuses. We concluded that in pigs testis cell proliferation, ovary development and histological organization was more pronounced during the final third of pregnancy. Fetal weight and size were strongly related to gonadal development, and can be used to estimate the histological characteristics of gonads.

Sperm Quality and Testicular Histomorphometry of Wistar Rats Supplemented with Extract and Fractions of Fruit of Tribulus terrestris L.

ABSTRACT The aim of this study was to assess the sperm quality and testicular histomorphometry of Wistar rats supplemented with extract and fractions of fruits of Tribulus terrestris L. The ethanolic extract was obtained by dynamic maceration of spray-dried fruit. This extract was fractionated by liquid-liquid partition, using increasing polarity solvents. Twenty male rats were separated in four groups, with five rats in each group. The control was supplemented with distilled water, while the others were daily given the ethanolic extract, hexanic or aqueous fraction soluble in methanol in a dose of 42 mg.kg-1.day-1 for 70 days. Sperm was obtained from the right epididymal tail for the analysis of motility, count, morphology and viability. The testicular weight of groups supplemented with ethanolic extract and aqueous fraction soluble in methanol was higher when compared to the control. The gonadosomatic index increased in the group supplemented with ethanolic extract. The nuclear, cytoplasmic and individual volume of Leydig cells increased in supplementation with hexanic and aqueous fractions soluble in methanol. It was concluded that the extract influenced the spermatogenesis, while hexanic and aqueous fractions soluble in methanol promoted the changes in the intertubular compartment. Therefore, Tribulus terrestris did not improve the sperm quality of the rats.