Effect of relaxin on in vitro fertilization of porcine oocytes

Porcine relaxin is a peptide hormone belonging to the insulin super family that has a variety of biological functions. The present experiment was designed to investigate the effects of relaxin on sperm function and on in vitro fertilization (IVF) of porcine oocytes. Porcine spermatozoa were washed, swum-up, and incubated for 1-4 h in mTALP medium supplemented with 0, 20 or 50 ng/ml porcine relaxin. Motility was determined by observing the type of forward movement of the spermatozoa, and acrosome status was evaluated by applying the triple staining technique. Immature oocytes were aspirated from antral follicles and matured in IVM medium (modified NCSU-37). Matured oocytes were co-cultured with spermatozoa in IVF medium (mTALP) supplemented with 0, 5, 10, 15 or 20 ng/ml relaxin. After 6 h of sperm-oocyte co-incubation, putative zygotes were cultured for 18 h in oocyte culture medium NCSU-37 and then assessed for the rates of monospermy, polyspermy, and male pronucleus formation after acetic orcein staining. Relaxin improved (P<0.05) sperm motility and increased the percentage of acrosome-reacted live spermatozoa during 1-4 h of incubation, although viability was not significantly improved. Significantly (P<0.05) the highest percentage of monospermic (31.7%) and lowest percentage of polyspermic (16.5%) fertilization was achieved from the sperm-oocyte co-culture group treated with 20 ng/ml relaxin as compared to other groups. The percentage of male pronucleus formation was significantly (P<0.05) greater in the 20 ng/ml relaxin-treated sperm-oocyte co-culture group than in the other groups. These results indicate that supplementation with relaxin is capable of improving sperm function and fertilization of porcine oocytes in vitro.     

In vitro capacitation of stallion spermatozoa assessed by the lysophosphatidylcholine-induced acrosome reaction and the penetration rate into in vitro-matured, zona-free mare oocytes

The purpose of this study was to evaluate the ability of various chemicals to induce capacitation of stallion spermatozoa using 2 different assay systems. In Experiment 1, freshly ejaculated spermatozoa were treated for 0, 3 and 6 h with 10 μ g/ml heparin, 0.5 mM hypotaurine or 5 mM caffeine, or were incubated for 0, 3 and 6 h following 1 min exposure to 0.1 μ M ionophore A23187. The acrosome reaction (AR) in the capacitated spermatozoa was induced by 15 min challenge with 100 μ g/ml lysophosphatidylcholine (LPC). In the BO/BSA-control medium (Brackett and Oliphant medium with 0.3% BSA), mean percentage of AR spermatozoa at 0 h was 30%, and the AR rates increased to 40 and 48% after 3 and 6 h incubation, respectively. There was no significant further increase of the AR rates in the spermatozoa treated with heparin (50% at 6 h) and hypotaurine (58% at 6 h) when compared to the control. Caffeine had a beneficial effect on inducing sperm capacitation after 3 and 6 h incubation (AR rates; 61 and 66%, respectively, P<0.01). Immediately after ionophore A23187 treatment, the AR rate increased to 56%, and reached 68 and 67% after 3 and 6 h incubation, respectively (P<0.01). Spermatozoal motility at any time points did not differ between control and any chemical treatment groups, except one treatment (ionophore; 3 h group).In Experiment 2, frozen-thawed spermatozoa were treated with 4 different chemicals as described above. Aliquot of spermatozoa was added to a microdrop of BO/BSA medium in which 6 to 10 in vitro-matured, zona-free mare oocytes were placed, and the oocytes were fixed and stained 20 h after insemination. The penetration rate by BO/BSA-treated spermatozoa was 76%, which was comparable to the results with heparin (73%), hypotaurine (78%) and caffeine (58%). In contrast, treatment of spermatozoa with ionophore A23187 gave a significantly lower penetration rate (30%) than the control value. Surprisingly these two experiments had different conclusions in assessing capacitation of stallion spermatozoa.     

Effects of 17beta-estradiol on in vitro maturation of pig oocytes in protein-free medium

The present study examined the effects of 17 beta-estradiol (E(2)) on in vitro maturation and subsequent in vitro fertilization of pig oocytes matured with or without cumulus cells. When E(2) (10 ng/ml) was added to the protein-free maturation medium, the proportions of cumulus-enclosed oocytes that underwent germinal vesicle breakdown and reached metaphase II were significantly reduced (P<0.05), and cumulus expansion was also significantly inhibited (P<0.05) compared with the control (no E(2) added). Although oocytes matured in the presence of E(2) were penetrated by sperm in vitro at the same level as the control, the incidences of male pronuclear (MPN) formation and activated oocytes were significantly lower (P<0.05) than the control. These inhibitory effects of E(2) were prevented when the medium was supplemented with E(2) together with its antagonist, ICI 182,780 (1 microg/ml), although the presence of the antagonist alone in the medium had no effect on the maturation and fertilization in vitro of oocytes. In cumulus-free oocytes, E(2) had no effect on nuclear maturation and penetration in vitro, but low MPN formation was observed in oocytes matured in the presence and absence of E(2). When cumulus-enclosed oocytes were cultured in the presence of progesterone (P(4); 600 ng/ml) alone or together with E(2), no significant differences in nuclear maturation, cumulus expansion or penetration in vitro were observed compared with control oocytes. The concentration of P(4) in maturation medium was significantly (P<0.01) lower when cumulus-enclosed oocytes were cultured for 44 h in the medium with E(2) than in medium without E(2). These results indicate that E(2) inhibits both nuclear and cytoplasmic maturation of cumulus-enclosed pig oocytes, and that this inhibition can be prevented by an E(2) antagonist or P(4). This E(2) inhibition may occur indirectly via the cumulus cells and inhibition of P(4) synthesis.     

In Vitro Development of Bovine Embryos after Fertilization using Semen from Different Donors

Contents: The aim of this study was to determine whether the semen donor and/or heparin concentration influences the rate of fertilization of bovine follicular oocytes and their subsequent embryonic development in vitro. Frozen-thawed semen from five highly fertile bulls was treated with one of four concentrations of heparin (0.5,1.0, 2.0 and 5.0 μg/ml) on a 5 x4 factorial basis in an IVM-NF programme. Zygotes/oocytes were cultured in frozen-thawed bovine oviduct cell-conditioned medium for 6 days. The use of semen from different bulls resulted in significantly (P < 0.001) different rates offertilization, as judged by cleavage rates of the oocytes at 72 h post insemination, and subsequent embryonic development through the'8-cell-block'(P < 0.05) in vitro. Development up to the morula/blastocyst stage, however, did not differ significantly (P = 0.06) among groups of oocytes fertilized with spermatozoa from different bulls. Heparin levels ranging from 0.5 to 5.0 μg/ml did not differ in their effect on in vitro fertilization as judged by the rate of normally cleaved oocytes (P = 0.14). The overall parthenogenetic division rate at 72 h post insemination was 12.4% and was not influenced by the heparin concentration. There was a linear relationship (P < 0.001) between fertility estimates based on AI and the estimates basedon the first cleavage following in vitro fertilization.     

Sperm penetration assay as an indicator of bull fertility

To predict the fertility of frozen-thawed bull spermatozoa, a sperm penetration assay (SPA) using zona-free hamster oocytes was optimized, and the assay results were compared with data from field fertility expressed as the non-return rate (NRR). To increase sperm penetration, the spermatozoa were pre-incubated and coincubated with oocytes in media containing various concentrations of heparin (0 to 50 μg/ml). Coincubation with 10 μg/ml heparin showed the highest sperm penetration (P<0.05); it is considered to be the optimized SPA method. Sperm fertility index values obtained from WSPA were significantly correlated with the historic average NRR of 46 bulls (P<0.01). To determine the normal range for SPA, we established the lower limits of the sperm fertility index and set the cut-off value at 2.55, at which point the NRR was more than 70%, using the receiver operating characteristic curve. The overall accuracy for the 46 bulls was 95.7% (44/46) for both the low and high NRR, with a sensitivity of 95.5% (21/22) and a specificity of 95.8%. This protocol would make it easier to discriminate bulls according to their sperm fertilizing ability.     

In Vitro and in Vivo Developmental Competence of Dromedary (Camelus dromedarius) Embryos Produced in Vitro Using Two Culture Systems (mKSOMaa and Oviductal Cells)

Development competence and pregnancy rate of in vitro-produced (IVP) dromedary embryos were studied in two culture systems: (i) semi-defined modified medium (mKSOMaa) and (ii) co-culture using camel epithelial oviducal cells. Five hundred and three cumulus-oocytes complexes (COCs) were selected, allowed to mature, fertilized and cultured in vitro (38.5 degrees C; 5% CO2, maximum humidity > 95%, with concentration of oxygen of 5% for semi-defined medium and 20% for co-culture cells). Maturation was accomplished by incubation in TCM-199 medium supplemented with 10% heat-treated foetal calf serum (FCS), 10 ng/ml epidermal growth factor, 1 microg/ml follicle-stimulating hormone, 1 microg/ml oestradiol and 500 microM cysteamine for 30 h. In vitro fertilization (IVF) was performed using fresh semen (0.5 x 10(6) spermatozoa/ml in modified TALP solution). Fertilized COCs were denuded by vortexing, then cultured in either mKSOMaa (10% heat-treated FCS was added 24 h post-IVF), under 5% O2 and 90% N2 (group 1; n = 249) or with dromedary epithelial oviducal cell monolayers in TCM-199 with 10% heat-treated FCS under 20% O2 (group 2; n = 254). The rate of cleavage was significant higher (p < 0.05) for group 1 (63%, 156/249) than for group 2 (51%, 130/254). No significant difference was found between the two groups in the rate of development to blastocyst (21% vs 16.5%) and their hatchability (21% vs 14%). Pregnancy rates were similar for the first 60 days. However, all pregnancies were lost after 60 days with the exception of two of six (33%) from recipients of hatched blastocysts from group 1. We conclude that both systems support in vitro production of dromedary embryos by in vitro maturation (IVM)/IVF of oocytes. However, embryos obtained by culture in the semi-defined medium (mKSOMaa) appear to have a better in vivo development ability.     

Nuclear and cytoplasmic maturation of bovine oocytes cultured with dbc AMP, FSH and hCG

The present investigation was undertaken to study the effect of addition of dbc AMP on bovine oocyte maturation and fertilization in vitro. The bovine oocytes isolated from 2–8 mm follicles were cultured for 26 h in TCM-199. The maturation rate (71.4 %) did not significantly increase after supplementation of the culture medium with dbc AMP (86.3 %.) or FSH + hCG (86.3 %). The in vitro fertilization rate of oocytes based on sperm penetration and presence of sperm tail in the ooplasm increased significantly in the dbc AMP (34.7 %) and the dbc AMP + FSH + hCG (33.9 %) treated groups when compared with untreated controls (17.9 %). However, dbc AMP treated oocytes were not able to secure the formation of male pronucleus 20 h after in vitro fertilization, while in oocytes matured in dbc AMP free medium both pronuclei were present in approximately 15 % of the penetrated oocytes. Also, the sperm head decondensation was blocked or slowed down by the dbc AMP treatment. It is concluded (1) that dbc AMP may improve the condition for the interaction of oocytes with spermatozoa, and (2) that the ooplasm of such dbc AMP treated oocytes apparently is not able to decandense the sperm head and transform it to the male pronucleus.     

Glutathione content of in vivo and in vitro matured canine oocytes collected from different reproductive stages

Glutathione (GSH) concentrations of oocytes are considered as an important marker of the cytoplasmic maturation. The present study was designed to compare GSH concentrations of in vivo and in vitro matured canine oocytes. In vivo matured oocytes were collected 72 hr after ovulation by flushing fallopian tubes after laparotomy. Ovaries were collected from bitches with different reproductive stages, and collected oocytes were divided into 2 groups according to the size viz. < 120 microm and > 120 microm in diameter and cultured for 72 hr in Tissue Culture Medium-199 supplemented with 10% FBS, 2.2 mg/ml sodium bicarbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution in the presence or absence of 50 microM beta-mercaptoethanol. GSH concentrations were determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. GSH concentrations of immature canine oocytes were 2.9 and 3.8, 3.5 and 6.8, and 3.1 and 6.5 pM/oocyte for < 120 microm and > 120 microm in diameter oocyte groups at anestrous, follicular and luteal stage, respectively (P<0.05). In vivo matured oocytes had significantly higher GSH concentrations compared with in vitro matured oocytes. The GSH content was 19.2 pM/oocyte for in vivo matured oocytes, while 4.1 to 8.1 and 5.7 to 13.2 pM/oocyte for in vitro matured oocytes cultured in the absence or presence of beta-mercaptoethanol, respectively (P<0.05). Presence of beta-mercaptoethanol increased GSH synthesis in canine oocytes cultured in vitro, and oocytes collected from follicular and luteal stage was superior to anestrus oocytes.     

Effect of low oxygen tension atmosphere and maturation media supplementation on nuclear maturation, cortical granules migration and sperm penetration in swine in vitro fertilization

The aim of this study was to evaluate the efficiency of low oxygen tension (5% CO(2) , 5% O(2) and 90% N(2) ) on in vitro oocyte maturation using defined media (0.1% polyvinyl alcohol - PVA) or 10% porcine follicular fluid (PFF)-supplemented media. To achieve this goal, oocytes were evaluated regarding cortical granules (GCs) migration, nuclear maturation and sperm penetration. Oocytes were in vitro matured under different conditions: 5% or 20% O(2) atmosphere and 0.1% PVA- or 10% PFF-supplemented media and evaluated at 0 and 44 h of maturation. To evaluate the migration of CGs and nuclear maturation, by confocal microscopy, oocytes were incubated with 100 μg of FITC-PNA/ml and 10 μg/ml of propidium iodide. To address sperm penetration, after maturation, in vitro fertilization for 6 h and in vitro culture for 18 h, zygotes were incubated with 10 mg/ml Hoechst 33342. Pronuclei and polar bodies were quantified using an epifluorescence microscope. Atmosphere conditions did not affect the CGs migration, but media supplementation did. Oocytes matured in 10% PFF media had a higher percentage of CGs in the oocyte periphery than oocytes matured in PVA-supplemented media. However, this fact did not have effect on in vitro sperm penetration levels. No effect of atmosphere conditions and media supplementation was observed on the rates of metaphase II oocytes. Therefore, the use of low oxygen tension in association with PVA maturation media does not improve the in vitro maturation system of porcine oocytes, because its use did not improve nuclear maturation, CGs migration and zygotes monospermic rates.     

In vitro Fertilizing Capacity of Deep Frozen Boar Semen Packaged in 0.5 and 5 ml Straws

The aim of this study was to evaluate the straw size effect used for freezing on the in vitro fertilizing capacity. Twenty-one ejaculates from seven fertile boars were frozen under controlled conditions in 0.5 and 5 ml straws. Thawed semen was compared to fresh semen. For fresh and thawed semen in 0.5 and 5 ml straws, the results were: 92.18, 77.38 and 79.04% sperm penetration; 80.68, 66.89 and 69.33% monospermy; 11.51, 10.49 and 9.74% polyspermy; 86.19, 47.14 and 47.02% motility and 75.52, 48.19 and 46.81% normal apical ridge (NAR), respectively. Analysis of variance and test of multiple comparisons showed that under the conditions employed, penetration, monospermy, motility and NAR were significantly reduced by freezing–thawing, but polyspermy was much less affected. The results obtained suggest that frozen boar semen is adequate for in vitro fertilization. In addition freezing in 5 ml straws did not have any detrimental effect on either penetration, monospermy, polyspermy, motility and NAR, in comparison with freezing in 0.5 ml straws.     

Evaluation of glucose as a cryoprotectant for boar semen

Fertility parameters of boar spermatozoa were evaluated in vitro, after freeze-thawing the semen in three different extenders containing permeable and non-permeable cryoprotectants: A (111.0 mM Tris, 31.4 mM citric acid, 185.0 mM glucose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G); B (200 mM Tris; 70.8 mM citric acid, 55.5 mM glucose, 20 per cent egg yolk, three per cent glycerol and 100 iu/ml penicillin G); C (200 mM Tris, 70.8 mM citric acid, 55.5 mM fructose, 20 per cent egg yolk, 3 per cent glycerol and 100 iu/ml penicillin G). The freeze-thawing techniques were the same for each extender. Eight ejaculates from four boars were obtained; the sperm-rich fraction of each ejaculate was extended in each of the three media at a final concentration of 400 x 106 sperm/ml, loaded into 0.5 ml straws and frozen at a rate of 30 degrees C/minute to -196 degrees C. The straws were thawed at 60 degrees C for eight seconds. Sperm motility, acrosomal integrity and in vitro sperm penetration through the zona pellucida of gilt oocytes matured in vitro were evaluated. The motility of unfrozen spermatozoa was 93.1 per cent compared with 60.7 per cent, 48.2 per cent and 35 per cent for sperm frozen in extenders A, B and C respectively; these values were all significantly different (P<0.05). There was no significant decline in sperm motility after incubation for 30 minutes in extender A, but there were significant decreases in sperm motility after 30 minutes of incubation in B and C. The percentage acrosomal integrities were 97.2 per cent for the control and 45.5 per cent, 30.3 per cent and 16.8 per cent for the frozen-thawed spermatozoa in extenders A, B and C respectively. The results of the in vitro penetration assay were 80.7 per cent when using control spermatozoa, and 42.2 per cent, 18.4 per cent and 3.3 per cent when using frozen-thawed spermatozoa in extenders A, B and C respectively     

In Vitro Fertilization and Development of Porcine Oocytes Matured in Follicular Fluid

This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 10⁶ cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium.     

Effects of sera and steroid hormones on development of bovine oocytes matured and fertilized in vitro and co-cultured with bovine oviduct epithelial cells

The present experiment was designed to identify possible effects of sera and steroid hormones added to a co-culture with bovine oviduct epithelial cells on embryonic development in vitro. Bovine oocytes were matured in vitro for 24 h and then fertilized in vitro using swim-up and heparin-treated, frozen-thawed spermatozoa. At 18 and 20 h after insemination, oocytes were cultured for 3 or 7 d in a co-culture system with bovine oviduct epithelial cells containing either fetal calf serum (FCS) or estrous cow serum (ECS) and one of six hormonal additions (none, 1 or 10 micrograms/ml estradiol [E]; 1 microgram/ml progesterone [P]; 1 microgram/ml E + P; and 10 micrograms/ml E + P). A total of 2,666 oocytes were cultured for 3 d and examined for cleavage. Of those, 2,280 oocytes were cultured up to 7 d for development to the late morula or blastocyst stage. Greatest cleavage rates for 2- to 8-cell and 8-cell stages were observed in FCS (71 and 24%) and ECS (66 and 23%) without steroid addition. For development into blastocysts, no serum effect was observed. Greatest rates for development into blastocysts were observed in FCS (14%) and ECS (16%) without steroid addition. These results indicate that addition of E and P at the doses and combinations tested did not enhance developmental capacity of in vitro fertilized bovine oocytes. Compared with FCS, ECS tended to increase cleavage rates and development into blastocysts.     

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 microM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm.     

Capacitation of Frozen Thawed Buffalo Bull (Bubalus bubalis) Spermatozoa with Higher Heparin Concentrations

The present study was conducted to examine the effect of high heparin concentration on capacitation of buffalo spermatozoa with a short incubation time. Frozen thawed spermatozoa from three buffalo bulls were pooled and treated with either 50, 100 or 200 microg/ml heparin for 30 min. Capacitation was evaluated by acrosome reaction of spermatozoa and in vitro fertilization rate (per cent cleavage rate, per cent cleavage index). Acrosome reaction was induced in heparin treated spermatozoa with calcium ionophore A23187 and staining was carried out with Coomassie G-250 to evaluate the response as compared with control (0 heparin + calcium ionophore). Significantly higher percentage of acrosome reaction (AR) spermatozoa was noted after heparin treatment (36.8-48.2%) as compared with control (8.1% ; p < 0.05) but differences among the three heparin concentrations were non-significant. However, a significantly higher in vitro fertilization rate was recorded in spermatozoa capacitated by 50 and 100 microg/ml heparin (80.4 and 75.9% cleavage rate, respectively) as compared with 200 microg/ml heparin (47.2% cleavage rate; p < 0.001). It is concluded that buffalo spermatozoa capacitated with 50-100 microg/ml heparin had significantly higher ability to improve in vitro fertilization rate in buffalo.     

Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa

The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sperm treatment groups. Semen was collected from 2 stallions with an artificial vagina and processed for each treatment. Treatment 1 (n = 10 mare cycles) consisted of fresh, nonsorted spermatozoa and Treatment 2 (n = 16 mare cycles) of fresh, flow sorted spermatozoa. Spermatozoa to be sorted were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Treatment 3 (n = 16 mare cycles) consisted of frozen/thawed nonsorted spermatozoa (frozen at 33.5 x 106 sperm/ml in 0.25 ml straws) and Treatment 4 (n = 15 mare cycles) of flow sorted frozen/thawed spermatozoa (frozen at 64.4 x 10(6) sperm/ml). Concentrations of sperm in both cryopreserved treatments were adjusted, based on predetermined average post-thaw motilities, so that each insemination contained approximately 5 x 10(6) motile spermatozoa. Hysteroscopic insemination of 5 x 10(6) motile spermatozoa in a volume of 230 microd was used for all treatments. Pregnancy was determined ultrasonographically 16 days postovulation. No differences were found (P>0.1) in the pregnancy rates for mares inseminated with fresh nonsorted (4/10 = 40.0%), fresh flow sorted (6/16 = 37.5%), frozen/thawed nonsorted (6/16 = 37.5%) and flow sorted frozen/thawed spermatozoa (2/15 = 133%). Pregnancy rates tended (P = 0.12) to be lower following insemination of frozen/thawed flow sorted spermatozoa. Further studies are needed with a larger number of mares to determine if fertility of flow sorted frozen/thawed spermatozoa can be improved.     

Effect of collection frequency on quantitative and qualitative characteristics of pigeon (Columba livia) semen

The aim was to estimate the optimal frequency of semen collection from pigeons in relation to ejaculate volume, sperm concentration, total spermatozoa in ejaculate and percentage of live morphologically normal cells. The study was carried out on 455 ejaculates collected from two groups of pigeons, each of 10 males (group I: meat-type breed; group II: fancy pigeon). The birds were selected and kept individually in cages under a natural photoperiod. A two-person technique was used for semen collection (lumbo-sacral and cloacal region massage). Semen was collected once, twice or three times per week. Colour, consistency and volume of ejaculates were evaluated macroscopically immediately after collection. Sperm concentration and total number of cells in the ejaculate were estimated after dilution with Ringer's solution. A live-dead stain technique (nigrosin-eosin) was used to determine the percentage of live and normal spermatozoa. Semen collected 3x/week was of high quality. The average volume of a single ejaculate was small (21 microl in group I and 19 microl in group II), but sperm concentration was high--1.58 x 10(9)/ml and 1.96 x 10(9)/ml, respectively. The mean number of spermatozoa per ejaculate was 30.48 x 10(6) in group I and 39.49 x 10(6) in group II. An increased percentage of live and normal spermatozoa in semen collected more frequently was also observed. Collecting pigeon semen 3x/week provides spermatozoa in larger amounts and of better quality than less frequent collections (1x/week or 2x/week) and is recommended for obtaining more insemination doses.     

表皮生长因子对水牛卵母细胞体外培养核质成熟的影响

为了探讨表皮生长因子（EGF）对水牛卵泡卵母细胞体外培养核质成熟的影响，在以TCM199为基础的成熟液中加入不同浓度的EGF（0、10、25、50、100 ng/ml），体外成熟培养24~26 h，观察第一极体（PB1）的排放；随后进行孤雌激活检测其分裂率、囊胚发育率、囊胚孵化率，并用Hoechst33342染色后计算囊胚的细胞数。结果发现添加EGF各组的卵母细胞第一极体排放率显著提高（P<0.05）；成熟液中添加25 ng/ml EGF时，卵裂率及囊胚发育率（分别为80.0%、44.8%）明显高于对照组（分别为69.3%、31.9%，P<0.05），但对囊胚的细胞数影响不大。EGF不仅促进水牛卵母细胞体外培养的核成熟，而且有利于卵母细胞的胞质成熟，其中EGF的最佳浓度为25 ng/ml。     

Production and lambing rate of blastocysts derived from in vitro matured oocytes after gonadotropin treatment of prepubertal ewes.

The aim of this study was to evaluate the effect of gonadotropin treatment on the in vitro maturation, blastocyst production, and developmental potential to term of oocytes collected from Sardinian neonatal and prepubertal ewes at 4 to 6 wk of age. Cumulus-oocyte complexes were recovered at 24 h after withdrawal of a 1/6th size progestagenated pessary from the donors, of which each received 120 IU FSH/LH and 400 IU PMSG in a single dose 36 h before sponge removal. Treated donors produced a greater (P<.01) number of oocytes per animal (86.2 +/-7.9) compared with slaughterhouse (untreated) prepubertal ewes (55.5+/-6.1) of the same age or with treated neonatal ewes (6.1+/-0.7) 10 d old. During oocyte maturation, there were no differences in the percentage of germinal vesicle break-down (78.08 vs. 74.24), metaphase I (89.13 vs. 87.18), and metaphase II (77.91 vs. 76.38) when evaluated after 8, 14, and 24 h of maturation, respectively, between oocytes from treated and slaughterhouse (untreated) prepubertal ewes. The embryo cleavage (71.1 vs. 73.7) and blastocyst rates (22.2 vs. 19.8) were similar in the treated and the untreated prepubertal ewes after transfer of in vitro matured oocytes into ligated oviducts of temporary recipients. The in vitro viability rates of vitrified blastocysts (81.2 vs. 76.9) and the in vivo survival rates (46.1 vs. 50.0) of embryos derived from in vitro matured and in vivo fertilized oocytes showed no difference. The data suggest that gonadotropin treatment increases oocyte production per animal but has no effect on oocyte quality because embryo production and lambing rates of blastocysts derived from in vitro matured oocytes were not markedly different from those derived from untreated prepubertal ewes of the same age.     

Single layer centrifugation-selected boar spermatozoa are capable of fertilization in vitro

Background

Good quality spermatozoa are important to achieve fertilization, viable embryos and offspring. Single Layer Centrifugation (SLC) through a colloid (Androcoll-P) selects good quality spermatozoa. However, it has not been established previously whether porcine spermatozoa selected by this method maintain their fertility.

Methods

The semen was prepared either by SLC or by standard centrifugation (control) and used for in vitro fertilization (IVF) at oocyte:spermatozoa ratios of 1:50; 1:100 and 1:300 (or 4 x 10³, 8 x 10³ and 24 x 10³ spermatozoa/ml) to evaluate their subsequent ability to generate blastocysts. In addition, sperm motility was assessed by computer assisted sperm motility analysis.

Results

Total and progressive motility were significantly higher in sperm samples prepared by SLC compared to uncentrifuged samples. Sperm binding ability, polyspermy, cleavage and blastocyst rates were affected by the oocyte:sperm ratio, but not by sperm treatment.

Conclusion

The use of SLC does not adversely affect the in vitro fertilizing and embryo-generating ability of the selected spermatozoa compared to their unselected counterparts, but further modifications in the IVF conditions would be needed to improve the monospermy in IVF systems. Since SLC did not appear to have a negative effect on sperm fertilizing ability, and may in fact select for spermatozoa with a greater potential for fertilization, an in vivo trial to determine the usefulness of this sperm preparation technique prior to artificial insemination is warranted.