G. Evans

Birth of a male lamb derived from an in vitro matured oocyte fertilised by intracytoplasmic injection of a single presumptive male sperm

The developmental competence of in vitro matured ovine oocytes, cytoplasmically injected with single male or female chromosome-bearing sperm, was investigated. Eighty-five unsorted, 92 ‘female-sorted’ and 74 ‘male-sorted’ ram sperm were injected into in vitro matured sheep oocytes and, two to four hours later, placed into the oviducts of 28 oestrous sheep. The sperm were separated according to sex by analysis of their DNA content with a flow cytometer. One pregnancy was diagnosed by ultrasound after 55 days and a 3 kg male lamb was born after 150 days gestation. This lamb was derived from an oocyte injected with ‘male-sorted’ sperm.

In vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing.

The effect of sex sorting and freeze-thawing on the viability and fertility of ram spermatozoa was investigated in the present study. Non-sorted (control) frozen-thawed spermatozoa had a higher motility and forwards progressive motility (FPM) than sorted frozen-thawed spermatozoa (60.9 +/- 2.9% v. 57.0 +/- 3.3% and 4.0 +/- 0.1 v. 3.5 +/- 0.1 FPM, respectively; P < 0.001) after incubation (6 h at 37 degrees C). Sorted and non-sorted (control) frozen-thawed spermatozoa had similar acrosome integrity (73.7 +/- 1.8% v. 75.2 +/- 2.1%, respectively) after thawing and incubation. A greater proportion of sorted spermatozoa displayed chlortetracycline staining patterns that were characteristic of capacitation (22.0 +/- 2.8%; P < 0.05) than non-sorted (control) spermatozoa (15.4 +/- 2.6% B pattern) before freezing. Overall, more sorted frozen-thawed spermatozoa showed patterns characteristic of being acrosome reacted (12.8 +/- 0.7%; P < 0.01) and less were uncapacitated (35.5 +/- 0.6%; P < 0.05) than non-sorted (control) frozen-thawed spermatozoa (7.7 +/- 0.8%; and 38.6 +/- 0.6% for AR and F pattern, respectively). Similar numbers of non-sorted (control) and sorted frozen-thawed spermatozoa migrated through artificial cervical mucus after 1 h (76.4 +/- 11.9 v. 73.9 +/- 11.9 spermatozoa, respectively). The distance travelled by the vanguard spermatozoon was also similar (56.9 +/- 7.8 v. 38.6 +/- 5.8 mm for control and sorted spermatozoa, respectively). Sorted and control frozen-thawed spermatozoa displayed a similar pattern of binding to, and release from, an oviduct epithelial cell monolayer (OECM), but sorted frozen-thawed spermatozoa were released more rapidly (P < 0.05) than non-sorted (control) frozen-thawed spermatozoa. The pregnancy rate was higher for ewes inseminated with 100 x 10(6) (commercial control) frozen-thawed spermatozoa (59%) than for 5, 10, 20 and 40 x 10(6) total sorted frozen-thawed spermatozoa (41% overall; P < 0.001). Insemination of 16 x 10(6) resulted in a higher pregnancy rate (31%) than 10(6) (17%; P < 0.05), but was similar to ewes that received 4 x 10(6) sorted frozen-thawed spermatozoa (24%). Time of insemination (54, 58 and 62 h after sponge removal) had no effect on pregnancy rate. Pregnancy in gonadotrophin-releasing hormone-treated ewes was affected by insemination dose (P < 0.05) but not sperm type (sorted and non-sorted) or ram. Pregnancy was higher after insemination of 40 x 10(6) than 5 or 20 x 10(6) non-sorted (control) or sorted frozen-thawed spermatozoa (70%, 33% and 35%, respectively; P < 0.05). Sorted frozen-thawed spermatozoa may have a shorter viability within the female tract than non-sorted frozen-thawed spermatozoa.

In vitro and in vivo developmental capacity of oocytes from prepubertal and adult sheep

Development to the blastocyst stage and survival following embryo transfer were assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The rates of maturation, fertilization and cleavage in vitro did not differ significantly between oocytes from prepubertal and adult sheep. The proportion of cleaved zygotes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal than for those from adult sheep (15.4% and 34.1% respectively). There were no differences in the pregnancy rate and number of lambs born following transfer of blastocyst stage embryos derived from prepubertal and adult sheep to adult recipients. These data show that embryos derived from prepubertal lamb oocytes have reduced developmental potential in vitro but, of those which do reach the blastocyst stage, they have equal capacity to develop to term as embryos derived from adult sheep.

Storage of semen and artificial insemination in deer

Methods of collection and freezing of semen of some deer species and aspects of controlled reproduction associated with the use of frozen-thawed semen by artificial insemination (AI) are discussed.

Effect of monosaccharides and disaccharides in Tris-based diluents on motility, acrosome integrity and fertility of pellet frozen ram spermatozoa.

Abstract In semen freezing diluents, sugars may be incorporated as metabolites, as cryoprotectants or to maintain diluent osmolality. This study examined the cryoprotective effects of mono- and disaccharides in Tris-glucose-egg yolk-glycerol based diluents on the post-thawing motility, acrosome integrity and fertility of pellet frozen ram spermatozoa. In Experiment 1, five concentrations of Tris (300, 250, 200, 150, 100 mM) and five concentrations of glucose (30, 75, 120, 165, 210 mM) were incorporated into freezing diluents. There was an interaction between Tris and glucose concentration on post-thawing motility of spermatozoa (P

Production of lambs of predetermined sex after the insemination of ewes with low numbers of frozen–thawed sorted X- or Y-chromosome-bearing spermatozoa

The fertilizing ability of sex-sorted frozen-thawed ram spermatozoa was assessed after insemination of mature Merino ewes at a synchronized oestrus. Ewes were inseminated into the uterus or utero-tubal junction (UTJ) with a total of 140 x 10(6) unsorted (control) or 2-4 x 10(6) sorted (X or Y) frozen-thawed ram spermatozoa 54 to 57 hours after removal of progestagen-impregnated pessaries and an injection of 400 IU of pregnant mare serum gonadotrophin (Folligon, Intervet). The spermatozoa were separated into X- and Y-chromosome-bearing spermatozoa after analysis with a modified high-speed cell sorter (SX MoFlo). The number of ewes pregnant after insemination with unsorted frozen-thawed spermatozoa was significantly higher (26/48; 54.3%) than for ewes inseminated with either X- (12/48; 25.0%) or Y-sorted spermatozoa (7/48; 14.6%) (P<0.05). Seventeen of the eighteen lambs produced by ewes inseminated with X-sorted spermatozoa were female (94.4%) and 8/8 lambs from ewes inseminated with Y-sorted spermatozoa were male (100%). The sex ratio of the lambs born to ewes inseminated with sex-sorted spermatozoa was significantly skewed from the 51.3% male and 48.7% female ratio in the control group (P<0.05). This study showed, for the first time, that lambs of predicted sex can be produced after insemination with low numbers of sex-sorted cryopreserved ram spermatozoa.

An interaction between feeding rate and season affects fertility of sows

Abstract Trials were conducted in two piggeries in the summer-autumn and winter-spring periods to determine the effects of rate of feeding and type of housing (group or individual stalls) on farrowing rate. Sows were fed a low (1.6–2.0 kg per sow per day), moderate (2.5 kg per sow per day) or high (> 3.6 kg per sow per day) rate for the first 4 weeks postmating and from then until farrowing fed at a moderate (2.5 or 3.2 kg per sow per day) rate. The low level of feeding in the summer-autumn was associated with increased numbers of delayed returns to oestrus and low farrowing rate ( 85%). Feeding sows at the high rate during the summer-autumn significantly improved the farrowing rate reducing the adverse effect of season. Litter size was not affected by feeding rate during early pregnancy. The seasonal effect on farrowing rate was not evident in individually stalled sows fed a moderate level during pregnancy. This study demonstrated effects on farrowing rate of interactions between rate of feeding and season and type of accommodation and season. Current practice restricting feed intake postmating, aimed at reducing embryonic mortality and increasing litter numbers, appeared to contribute substantially to the decrease in farrowing rate commonly observed in summer-autumn. The optimal level of feeding to minimise the effect of season on fertility has yet to be defined. Accommodation of sows in individual stalls, rather than in groups, removed the seasonal effect on fertility.

Ovarian and endocrine responses of Merino ewes to treatment with PMSG and/or FSH-P

An experiment was conducted to investigate the effects of different superovulatory regimes, including pregnant mare serum gonadotrophin (PMSG) and follicle-stimulating hormone (FSH-P) on steroidogenesis, oestrus, ovulation and premature luteal regression in sheep. Thirty mature Merino ewes were treated in the breeding season with intravaginal progestagen pessaries and 1200 IU PMSG (Group 1), 15 mg FSH-P (Group 2) or a combination of 700 IU PMSG and 11 mg FSH-P (PMSG/FSH-P; Group 3) to induce superovulation. After treatment with FSH-P alone, the incidence of oestrus was lower than after treatment with PMSG alone (6/10 vs. 10/10, P < 0.05) and the incidence of ovulation was lower than after treatment including PMSG (7/10 vs. 20/20, P < 0.05). However, there were no differences in the mean time to onset of oestrus (24.7 ± 2.3 h after pessary withdrawal) or the time to onset of ovulation (48–54 h after pessary withdrawal) between the treatment groups. The total ovarian response (corpora lutea + large follicles) was higher (P < 0.05) following stimulation with PMSG alone (17.1 ± 1.9) than with FSH-P (8.3 ± 12.5). However, the numbers of corpora lutea present on Day 6 (Day 0 = pessary withdrawal) were not affected by the exogenous gonadotrophin treatment (PMSG, 11.4 ± 1.8; FSH-P, 7.2 ± 2.5; PMSG/FSH-P, 11.1 ± 2.1; not significant). The mean pre-ovulatory peripheral plasma peak level of oestradiol-17β (E2) per follicle was higher following treatment with PMSG than with either of the other two regimes (PMSG, 23.2 ± 0.8 pg ml−1; FSHP, 10.8 ± 0.6 pg ml−1; PMSG/FSH-P, 13.9 ± 0.4 pg ml−1; P < 0.05). Moreover, E2 peaked earlier in the ewes treated with PMSG than with FSH-P or PMSG/FSH-P (7.6 ± 2.1 h vs. 33.7 ± 1.5 h and 24.8 ± 0.5 h respectively; P < 0.01). There were no differences detected in the peak levels of luteinizing hormone (LH) (8.6 ± 1.1 ng ml−1) or the mean time to LH peak (32 ± 1.4 h) between the ewes treated with the different exogenous gonadotrophins. Three ewes (one treated with PMSG alone and two with PMSG/FSH) had premature luteal regression by Day 6. In each case, a secondary peak of E2 secretion was observed 36–44 h after pessary withdrawal. These ewes each displayed a second oestrus on Days 5–6 and a mild superovulatory response at this oestrus. It is concluded that in sheep PMSG is highly steroidogenic in comparison with FSH-P. A combination of moderate doses of PMSG and FSH-P is a suitable superovulation regime for Merino ewes as it induces a consistently high superovulatory response without the secretion of excessive E2 levels. Perturbation of the periovulatory endocrine events may result in premature luteal regression.

Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization.

More abnormal fertilization has been found in sheep oocytes after intracytoplasmic sperm injection (ICSI) than after in vitro fertilization (IVF). Although the birth of a normal lamb has been reported, the efficiency of blastocyst production is low. We therefore evaluated the cleavage, development and viability of sheep embryos obtained from ICSI, IVF and sham injection. In vitro matured oocytes either injected or inseminated with spermatozoa were assessed for cleavage 1 and 4 d after injection or insemination, and for development to blastocyst after 7 d of culture. A total of 699 oocytes was injected (ICSI); 198 (30.6%) were activated and 55 (8.5%) developed to the blastocyst stage. Of the 17 recipient ewes with 1, 2, 3 or 4 embryos, 15 (88.2%) were pregnant on Day 18; of these 17 recipients, 7 (41.1%) and 6 (35.2%) ewes remained pregnant on Days 45 and 110, respectively. Two normal lambs were born, one ewe died on Day 110 with 2 normal male fetuses, another ewe aborted on Day 90 and 4 pregnancies were maintained. A total of 517 oocytes was inseminated (IVF); 296 (62%) were activated and 90 (18.8%) reached the blastocyst stage. A total of 19 ewes received 1, 2, 3 or 4 embryos; of these, 13 (68.4%) were pregnant on Day 18, 8 (42.1%) ewes remained pregnant on each of Days 45 and 110. Three ewes delivered 5 lambs. Five pregnancies were maintained. A total of 156 oocytes was sham injected, 38 (24.3%) were activated and no blatocysts were obtained after culture. The results of this study showed that blastocysts obtained after ICSI are potentially viable and are not a result of parthenogenesis.

Effect of chronic treatment with a GnRH agonist (Goserelin) on LH secretion and early pregnancy in gilts

Abstract A slow release implant containing the gonadotrophin-releasing hormone (GnRH) analogue Goserelin acetate (D-Ser[Bu t ] 6 , Azgly-NH 2 10 ) a for GnRH receptor down-regulation in the pituitary was used in gilts, to determine what effects elimination of episodic luteinising hormone (LH) peaks may have on progesterone secretion in the oestrus cycle and in early pregnancy, and on the establishment and maintenance of pregnancies. Groups of non-mated and mated gilts were implanted with the GnRH agonist on day of oestrus (Day 0) and on Days 0, 14, 21 and 29 of pregnancy. The GnRH implant caused increased plasma LH concentrations for 38 h with a peak occurring 14 h post treatment, after which there was a prolonged period during which episodic LH release was abolished. A transient increase in progesterone concentration followed GnRH implant insertion and a steep decline on the day of abortion or the 2 days preceding it was seen in the majority of GnRH agonist treated gilts. In the cyclic gilts, the implant did not alter the pattern of progesterone secretion. In all gilts implanted before Day 29, pregnancy failed. However, in 50% of the gilts implanted on Day 29, pregnancy was maintained. In conclusion, these results indicate that episodic LH peaks play an important role in the establishment of pregnancy and are essential for the maintenance of pregnancy before Day 29.