Artificial insemination (AI) is poorly developed in camelids owing to the difficulty in collectin... more Artificial insemination (AI) is poorly developed in camelids owing to the difficulty in collecting high quality semen and the highly viscous nature of the semen. Semen collected by artificial vagina (AV) is often of low quality and must be improved before any further development of AI technology can occur. The present study investigated the effects of adding a cervix-like stricture to the AV, presence of females, collecting semen into Androhep, skim-milk or Tris diluents, and catalase supplementation (0, 100, 200 or 600 units/ml) of Tris diluent on alpaca semen quality parameters. The addition of a cervix-like stricture increased mating length (p < 0.05), whilst the presence of females during semen collection did not improve semen quality parameters (p > 0.05). Collection of semen into Tris diluent improved sperm motility (58.0 +/- 11.9%) compared with the control (34.0 +/- 10.8%; p < 0.05), Androhep (33.5 +/- 10.7%) and skim-milk diluents (28.2 +/- 10.4%). Semen viscosity was reduced by collection into Androhep (4.6 +/- 1.7 mm) and skim-milk diluents (3.6 +/- 1.3 mm) compared with Tris diluent (5.7 +/- 2.1 mm) and no collection medium (9.3 +/- 3.5 mm; p < 0.05). Tris diluent supplemented with 100, 200 or 600 units/ml catalase increased semen viscosity (5.0 +/- 3.2 and 4.9 +/- 3.2 mm). Collection of alpaca semen by AV into Tris diluent increased semen quality facilitating further development of AI technology in alpacas.
In vitro matured adult (Experiment 1) and prepubertal (Experiment 2) ewe oocytes were co-incubate... more 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.
The in vitro and in vivo developmental capabilities and kinetics of in vitro development of embry... more The in vitro and in vivo developmental capabilities and kinetics of in vitro development of embryos derived from adult ewes and from unstimulated (16- to 24-week-old) and hormone-stimulated prepubertal (3- to 5-week-old) ewes were assessed. Cleavage was lower for hormone-stimulated (617/1025; 60.2%) than unstimulated prepubertal (117/169; 69.2%) and adult ewe oocytes (184/267; 68.9%; P < 0.05). Blastocyst formation by Day 7 (from zygotes) was similar for unstimulated (45/117; 38.5%), hormone-stimulated prepubertal (229/617; 37.1%) and adult ewes (101/184; 54.9%). Blastocysts derived from hormone-stimulated prepubertal ewes developed mainly on day 7, compared with Day 6 for adult and unstimulated prepubertal ewes. Pregnancy rates (day 60) and embryonic loss (between Days 20 and 60) did not differ after transfer to adult recipient ewes of adult, unstimulated and hormone-stimulated prepubertal-derived fresh or frozen-thawed embryos. The number of lambs born as a proportion of embryos transferred did not differ for fresh and frozen embryos derived from adult ewes (3/16; 18.8% and 1/12; 8.3%, respectively) and unstimulated prepubertal lambs (2/6; 33.3%, and 1/10; 10.0%, respectively), but was higher for fresh than frozen embryos from hormone-stimulated prepubertal ewes (7/16; 43.8%, and 2/14; 14.3%, respectively; P < 0.05). There were high rates of in vitro and in vivo development of oocytes from 3- to 5-week-old lambs, but in vitro development was lower than with oocytes from adult ewes. However, the speed of embryonic development in vitro and the in vivo development of fresh and frozen embryos were similar to those derived from adult and unstimulated prepubertal ewes. The present results were an improvement in the efficiency of producing embryos and offspring from hormone-stimulated 3- to 5-week-old lambs.
Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prep... more Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p <0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p <0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p >0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p >0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p >0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.
Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment ... more Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment 1) and response to stimulation (Experiment 2) on the in vitro production of embryos from prepubertal lambs aged 3-4 and 6-7 weeks of age. For 3-4-week-old lambs, hormone stimulation increased the number of follicles (29.9 +/- 15.3 v. 70.6 +/- 8.2), oocytes per ovary (18.3 +/- 6.3 v. 39.3 +/- 5.8) and oocyte development to the blastocyst stage (0/192 (0.0%) v. 115/661 (17.4%); P < 0.05). Lamb age (3-4 v. 6-7 weeks old) increased oocyte development to the blastocyst stage (115/661 (17.4%) v. 120/562 (21.4%) respectively). In Experiment 2, hormone-stimulated lambs (3-4 and 6-7 weeks old) were divided into low, medium or high responders based on the number of ovarian follicles (<20, 20-50 and >100 follicles per ovary respectively). The response to hormone stimulation did not affect oocyte recovery rate, but the number of oocytes suitable for culture was increased for high-responding...
The production of embryos from prepubertal lambs is inefficient, partly resulting from the low de... more The production of embryos from prepubertal lambs is inefficient, partly resulting from the low developmental competence of prepubertal lamb oocytes, and partly because a high proportion of lambs fail to respond to hormone stimulation. The development of a hormone stimulation regimen that all lambs respond to would increase the efficiency of breeding from prepubertal animals. Using a hormone stimulation regimen consisting of oestradiol benzoate (50 microg), a norgestomet implant (1.5 mg), pregnant mare serum gonadotrophin (400 IU) and follicle stimulating hormone (130 mg) all lambs (n = 19) responded to hormone stimulation. Uterine and ovarian weight ranged from 2.8 to 7.2 g (11.8 +/- 0.7 g) and from 1.7 to 54.1 (12.5 +/- 2.9 g), respectively. The number of ovarian follicles and oocytes recovered ranged from 20.0 to 500.0 (118.2 +/- 29.2) and from 13.0 to 455.0 (82.0 +/- 24.2), respectively, and oocytes suitable for in vitro production were obtained from all 19 lambs. Uterine weight ...
Sperm-sexing has been used to produce embryos and offspring of a pre-determined sex in a number o... more Sperm-sexing has been used to produce embryos and offspring of a pre-determined sex in a number of species. However, the fertility of sex-sorted sperm is reduced and the full effects of sperm-sexing remain to be elucidated. The purpose of the present study was to investigate the potential effects of sex-sorted sperm on mRNA expression patterns of developmentally important genes employing in vitro produced bovine embryos. Bovine embryos were produced in vitro with unsorted and sex-sorted sperm and mRNA expression patterns were determined for glucose-3 transporter (Glut-3), glucose-6-phosphate dehydrogenase (G6PD), X-inactive specific transcript (X-ist) and Heat shock protein 70.1 (Hsp) using semi-quantitative endpoint reverse transcriptase-PCR in male and female, day-7 and 8 embryos. The relative abundance (RA) of Glut-3 was higher for day-7 male than female embryos, and day-7 embryos derived from unsorted compared with sex-sorted sperm. The RA of G6PD was higher for embryos derived from unsorted than sex-sorted sperm, and for day-8 female compared with male embryos. The RA of Xist was higher for female than male embryos, and for day-7 female embryos derived from unsorted than sex-sorted sperm. Hsp RA was higher for female compared with male embryos, was similar for day-7 and 8 embryos, and unsorted and sex-sorted sperm derived embryos. These results demonstrate differential expression of developmentally important genes between male and female embryos, and embryos derived from unsorted and sex-sorted sperm.
Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment ... more Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment 1) and response to stimulation (Experiment 2) on the in vitro production of embryos from prepubertal lambs aged 3-4 and 6-7 weeks of age. For 3-4-week-old lambs, hormone stimulation increased the number of follicles (29.9 +/- 15.3 v. 70.6 +/- 8.2), oocytes per ovary (18.3 +/- 6.3 v. 39.3 +/- 5.8) and oocyte development to the blastocyst stage (0/192 (0.0%) v. 115/661 (17.4%); P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Lamb age (3-4 v. 6-7 weeks old) increased oocyte development to the blastocyst stage (115/661 (17.4%) v. 120/562 (21.4%) respectively). In Experiment 2, hormone-stimulated lambs (3-4 and 6-7 weeks old) were divided into low, medium or high responders based on the number of ovarian follicles (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;20, 20-50 and &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;100 follicles per ovary respectively). The response to hormone stimulation did not affect oocyte recovery rate, but the number of oocytes suitable for culture was increased for high-responding 3-4-week-old lambs only (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Oocyte development to the blastocyst stage was not affected by response to stimulation for 3-4-week-old lambs (15.2-25.6%; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05), but was reduced for high (6.7%) compared with low (19.5%) and medium (30.9%) responding 6-7-week-old lambs (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). These results demonstrate that the production of embryos from prepubertal lambs is increased by hormone stimulation and lamb age and the response to stimulation does not affect embryo production from 3-4-week-old lambs, although by 6-7 weeks of age a high response to stimulation reduces blastocyst formation.
Epididymal spermatozoa were harvested from male alpacas and frozen after extension and cooling to... more Epididymal spermatozoa were harvested from male alpacas and frozen after extension and cooling to 4 degrees C in citrate-, Tris- and lactose-based diluents (Experiment 1) and as pellets in 0.25- and 0.5-mL straws on either dry ice or over liquid nitrogen vapour (Experiment 2) to determine the effects diluents and packaging on their motility and acrosome integrity. In Experiment 1, sperm motility was higher after cooling to 4 degrees C and after freeze-thawing (0 but not 3 h post-thaw) for spermatozoa extended in the lactose- than the citrate- or Tris-based diluent (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Post-thaw acrosome integrity after cooling to 4 degrees C and post-thaw (0 h) was reduced for spermatozoa frozen in citrate- compared with lactose- or Tris-based diluents, but was similar for all groups 3 h after thawing. In Experiment 2, sperm motility immediately after thawing was higher for pellet freezing than for 0.25- or 0.5-mL straws on dry ice or liquid nitrogen vapour (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), although by 3 h post-thaw motility was similar for pellets and straws (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05). Acrosome integrity was similar for all groups immediately after thawing and 3 h post-thaw. Cryopreservation of epididymal alpaca spermatozoa is feasible, with retained motility and acrosome integrity post-thaw. Freezing as pellets in a lactose-based diluent is recommended.
Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prep... more Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p &lt;0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p &lt;0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p &gt;0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p &gt;0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p &gt;0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.
Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with un... more Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with unfrozen sperm, and after surgical insemination with sex-sorted frozen-thawed sperm. The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a pre-determined sex and that these embryos could be successfully non-surgically transferred. Oocytes were matured in vitro, fertilised with either unsorted or sex-sorted frozen-thawed sperm and cultured until the eight-cell stage. These embryos were then transferred to recipients (n = 7) non-surgically (n = 70 embryos per sow). Oocyte cleavage was similar between sex-sorted (1538/5044; 30.5%) and unsorted (216/756; 28.6%) frozen-thawed sperm, and PCR sex-determination of the embryos confirmed that they were of the predicted sex (n = 16). Delayed return to oestrus (&gt;23 days) was observed in five recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound on Day 18 in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine ET protocols are required to enable development to term.
Artificial insemination (AI) is poorly developed in camelids owing to the difficulty in collectin... more Artificial insemination (AI) is poorly developed in camelids owing to the difficulty in collecting high quality semen and the highly viscous nature of the semen. Semen collected by artificial vagina (AV) is often of low quality and must be improved before any further development of AI technology can occur. The present study investigated the effects of adding a cervix-like stricture to the AV, presence of females, collecting semen into Androhep, skim-milk or Tris diluents, and catalase supplementation (0, 100, 200 or 600 units/ml) of Tris diluent on alpaca semen quality parameters. The addition of a cervix-like stricture increased mating length (p &amp;amp;lt; 0.05), whilst the presence of females during semen collection did not improve semen quality parameters (p &amp;amp;gt; 0.05). Collection of semen into Tris diluent improved sperm motility (58.0 +/- 11.9%) compared with the control (34.0 +/- 10.8%; p &amp;amp;lt; 0.05), Androhep (33.5 +/- 10.7%) and skim-milk diluents (28.2 +/- 10.4%). Semen viscosity was reduced by collection into Androhep (4.6 +/- 1.7 mm) and skim-milk diluents (3.6 +/- 1.3 mm) compared with Tris diluent (5.7 +/- 2.1 mm) and no collection medium (9.3 +/- 3.5 mm; p &amp;amp;lt; 0.05). Tris diluent supplemented with 100, 200 or 600 units/ml catalase increased semen viscosity (5.0 +/- 3.2 and 4.9 +/- 3.2 mm). Collection of alpaca semen by AV into Tris diluent increased semen quality facilitating further development of AI technology in alpacas.
In vitro matured adult (Experiment 1) and prepubertal (Experiment 2) ewe oocytes were co-incubate... more 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 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.
The in vitro and in vivo developmental capabilities and kinetics of in vitro development of embry... more The in vitro and in vivo developmental capabilities and kinetics of in vitro development of embryos derived from adult ewes and from unstimulated (16- to 24-week-old) and hormone-stimulated prepubertal (3- to 5-week-old) ewes were assessed. Cleavage was lower for hormone-stimulated (617/1025; 60.2%) than unstimulated prepubertal (117/169; 69.2%) and adult ewe oocytes (184/267; 68.9%; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Blastocyst formation by Day 7 (from zygotes) was similar for unstimulated (45/117; 38.5%), hormone-stimulated prepubertal (229/617; 37.1%) and adult ewes (101/184; 54.9%). Blastocysts derived from hormone-stimulated prepubertal ewes developed mainly on day 7, compared with Day 6 for adult and unstimulated prepubertal ewes. Pregnancy rates (day 60) and embryonic loss (between Days 20 and 60) did not differ after transfer to adult recipient ewes of adult, unstimulated and hormone-stimulated prepubertal-derived fresh or frozen-thawed embryos. The number of lambs born as a proportion of embryos transferred did not differ for fresh and frozen embryos derived from adult ewes (3/16; 18.8% and 1/12; 8.3%, respectively) and unstimulated prepubertal lambs (2/6; 33.3%, and 1/10; 10.0%, respectively), but was higher for fresh than frozen embryos from hormone-stimulated prepubertal ewes (7/16; 43.8%, and 2/14; 14.3%, respectively; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). There were high rates of in vitro and in vivo development of oocytes from 3- to 5-week-old lambs, but in vitro development was lower than with oocytes from adult ewes. However, the speed of embryonic development in vitro and the in vivo development of fresh and frozen embryos were similar to those derived from adult and unstimulated prepubertal ewes. The present results were an improvement in the efficiency of producing embryos and offspring from hormone-stimulated 3- to 5-week-old lambs.
Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prep... more Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p &lt;0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p &lt;0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p &gt;0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p &gt;0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p &gt;0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.
Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment ... more Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment 1) and response to stimulation (Experiment 2) on the in vitro production of embryos from prepubertal lambs aged 3-4 and 6-7 weeks of age. For 3-4-week-old lambs, hormone stimulation increased the number of follicles (29.9 +/- 15.3 v. 70.6 +/- 8.2), oocytes per ovary (18.3 +/- 6.3 v. 39.3 +/- 5.8) and oocyte development to the blastocyst stage (0/192 (0.0%) v. 115/661 (17.4%); P < 0.05). Lamb age (3-4 v. 6-7 weeks old) increased oocyte development to the blastocyst stage (115/661 (17.4%) v. 120/562 (21.4%) respectively). In Experiment 2, hormone-stimulated lambs (3-4 and 6-7 weeks old) were divided into low, medium or high responders based on the number of ovarian follicles (<20, 20-50 and >100 follicles per ovary respectively). The response to hormone stimulation did not affect oocyte recovery rate, but the number of oocytes suitable for culture was increased for high-responding...
The production of embryos from prepubertal lambs is inefficient, partly resulting from the low de... more The production of embryos from prepubertal lambs is inefficient, partly resulting from the low developmental competence of prepubertal lamb oocytes, and partly because a high proportion of lambs fail to respond to hormone stimulation. The development of a hormone stimulation regimen that all lambs respond to would increase the efficiency of breeding from prepubertal animals. Using a hormone stimulation regimen consisting of oestradiol benzoate (50 microg), a norgestomet implant (1.5 mg), pregnant mare serum gonadotrophin (400 IU) and follicle stimulating hormone (130 mg) all lambs (n = 19) responded to hormone stimulation. Uterine and ovarian weight ranged from 2.8 to 7.2 g (11.8 +/- 0.7 g) and from 1.7 to 54.1 (12.5 +/- 2.9 g), respectively. The number of ovarian follicles and oocytes recovered ranged from 20.0 to 500.0 (118.2 +/- 29.2) and from 13.0 to 455.0 (82.0 +/- 24.2), respectively, and oocytes suitable for in vitro production were obtained from all 19 lambs. Uterine weight ...
Sperm-sexing has been used to produce embryos and offspring of a pre-determined sex in a number o... more Sperm-sexing has been used to produce embryos and offspring of a pre-determined sex in a number of species. However, the fertility of sex-sorted sperm is reduced and the full effects of sperm-sexing remain to be elucidated. The purpose of the present study was to investigate the potential effects of sex-sorted sperm on mRNA expression patterns of developmentally important genes employing in vitro produced bovine embryos. Bovine embryos were produced in vitro with unsorted and sex-sorted sperm and mRNA expression patterns were determined for glucose-3 transporter (Glut-3), glucose-6-phosphate dehydrogenase (G6PD), X-inactive specific transcript (X-ist) and Heat shock protein 70.1 (Hsp) using semi-quantitative endpoint reverse transcriptase-PCR in male and female, day-7 and 8 embryos. The relative abundance (RA) of Glut-3 was higher for day-7 male than female embryos, and day-7 embryos derived from unsorted compared with sex-sorted sperm. The RA of G6PD was higher for embryos derived from unsorted than sex-sorted sperm, and for day-8 female compared with male embryos. The RA of Xist was higher for female than male embryos, and for day-7 female embryos derived from unsorted than sex-sorted sperm. Hsp RA was higher for female compared with male embryos, was similar for day-7 and 8 embryos, and unsorted and sex-sorted sperm derived embryos. These results demonstrate differential expression of developmentally important genes between male and female embryos, and embryos derived from unsorted and sex-sorted sperm.
Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment ... more Experiments were conducted to determine the effects of lamb age, hormone stimulation (Experiment 1) and response to stimulation (Experiment 2) on the in vitro production of embryos from prepubertal lambs aged 3-4 and 6-7 weeks of age. For 3-4-week-old lambs, hormone stimulation increased the number of follicles (29.9 +/- 15.3 v. 70.6 +/- 8.2), oocytes per ovary (18.3 +/- 6.3 v. 39.3 +/- 5.8) and oocyte development to the blastocyst stage (0/192 (0.0%) v. 115/661 (17.4%); P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Lamb age (3-4 v. 6-7 weeks old) increased oocyte development to the blastocyst stage (115/661 (17.4%) v. 120/562 (21.4%) respectively). In Experiment 2, hormone-stimulated lambs (3-4 and 6-7 weeks old) were divided into low, medium or high responders based on the number of ovarian follicles (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;20, 20-50 and &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;100 follicles per ovary respectively). The response to hormone stimulation did not affect oocyte recovery rate, but the number of oocytes suitable for culture was increased for high-responding 3-4-week-old lambs only (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Oocyte development to the blastocyst stage was not affected by response to stimulation for 3-4-week-old lambs (15.2-25.6%; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05), but was reduced for high (6.7%) compared with low (19.5%) and medium (30.9%) responding 6-7-week-old lambs (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). These results demonstrate that the production of embryos from prepubertal lambs is increased by hormone stimulation and lamb age and the response to stimulation does not affect embryo production from 3-4-week-old lambs, although by 6-7 weeks of age a high response to stimulation reduces blastocyst formation.
Epididymal spermatozoa were harvested from male alpacas and frozen after extension and cooling to... more Epididymal spermatozoa were harvested from male alpacas and frozen after extension and cooling to 4 degrees C in citrate-, Tris- and lactose-based diluents (Experiment 1) and as pellets in 0.25- and 0.5-mL straws on either dry ice or over liquid nitrogen vapour (Experiment 2) to determine the effects diluents and packaging on their motility and acrosome integrity. In Experiment 1, sperm motility was higher after cooling to 4 degrees C and after freeze-thawing (0 but not 3 h post-thaw) for spermatozoa extended in the lactose- than the citrate- or Tris-based diluent (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Post-thaw acrosome integrity after cooling to 4 degrees C and post-thaw (0 h) was reduced for spermatozoa frozen in citrate- compared with lactose- or Tris-based diluents, but was similar for all groups 3 h after thawing. In Experiment 2, sperm motility immediately after thawing was higher for pellet freezing than for 0.25- or 0.5-mL straws on dry ice or liquid nitrogen vapour (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), although by 3 h post-thaw motility was similar for pellets and straws (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05). Acrosome integrity was similar for all groups immediately after thawing and 3 h post-thaw. Cryopreservation of epididymal alpaca spermatozoa is feasible, with retained motility and acrosome integrity post-thaw. Freezing as pellets in a lactose-based diluent is recommended.
Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prep... more Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p &lt;0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p &lt;0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p &gt;0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p &gt;0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p &gt;0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.
Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with un... more Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with unfrozen sperm, and after surgical insemination with sex-sorted frozen-thawed sperm. The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a pre-determined sex and that these embryos could be successfully non-surgically transferred. Oocytes were matured in vitro, fertilised with either unsorted or sex-sorted frozen-thawed sperm and cultured until the eight-cell stage. These embryos were then transferred to recipients (n = 7) non-surgically (n = 70 embryos per sow). Oocyte cleavage was similar between sex-sorted (1538/5044; 30.5%) and unsorted (216/756; 28.6%) frozen-thawed sperm, and PCR sex-determination of the embryos confirmed that they were of the predicted sex (n = 16). Delayed return to oestrus (&gt;23 days) was observed in five recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound on Day 18 in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine ET protocols are required to enable development to term.
Breeding from prepubertal females, known as juvenile in vitro embryo transfer (JIVET), reduces th... more Breeding from prepubertal females, known as juvenile in vitro embryo transfer (JIVET), reduces the generation interval and increases the rate of genetic gain in animal breeding programs. While the birth of the first lambs from prepubertal ewes occurred nearly 30 years ago; and there is considerable interest in the commercialization of this technology, its efficiency remains too low. The advent of in vitro production (IVP) of embryo resulted in the more widespread use of JIVET. Morphologic and metabolic differences coupled with reduced in vitro and in vivo development of oocytes derived from prepubertal animals have been reported. Research has been undertaken to optimize donor selection and hormone stimulation methods in an attempt to reduce the variability and increase the proportion of donors responding to hormone stimulation and increase oocyte developmental competence. Yet, this variation persists and the development of oocytes and embryos from prepubertal animals remains reduced when compared with adults. Recent improvements to JIVET, resulting from a modified hormone stimulation regime, have eliminated the failure of donors to respond to hormone stimulation, and increased both the number and developmental competence of oocytes harvested from very young prepubertal lambs. This increased efficiency has facilitated the incorporation of other reproductive technologies such as sperm sexing with JIVET, resulting in the birth of lambs of a pre-determined sex from prepubertal lambs. Increased rates of genetic gain in sheep breeding programs can be achieved by combining sexed sperm with oocytes obtained from lambs as young as 3-4 weeks of age. Continued increases in the efficiency of JIVET resulting from further improvements to hormone stimulation regimes and an increased understanding of the differences between oocytes from adult and prepubertal animals will result in the commercialization of this technology.
Liquid storage of alpaca semen was investigated by assessing the motility and acrosome integrity ... more Liquid storage of alpaca semen was investigated by assessing the motility and acrosome integrity of sperm at 24, 48 and 72 h after i) dilution 1:4 (v/v) with Androhep®, Biladyl®, Lactose, Salamon's and Triladyl® diluents and liquid storage at 4 or 15°C (Experiment 1), ii) dilution 1:1, 1:2, or 1:4 (v/v) with Biladyl® and storage at 4 or 15°C (Experiment 2) and iii) dilution of epididymal sperm with Biladyl® and storage at 4°C for 24 h. In Experiment 1, sperm motility was higher after storage in Biladyl® compared with other diluents (P<0.05), and sperm motility was similar for Lactose and Triladyl® diluents but lower for Androhep® and Salamon's diluents (P<0.05). Sperm motility was similar at 24 and 48 h after storage at 4 and 15°C, but higher at 72 h after storage at 4°C (P<0.05). Acrosome integrity did not decline until 72 h of liquid storage. In Experiment 2, sperm motility was higher after dilution 1:4 than 1:1 or 1:2 with Biladyl® (P<0.05). Acrosome integrity...
ABSTRACT Juvenile barramundi were fed one of six diets containing differing docosahexaenoic acid ... more ABSTRACT Juvenile barramundi were fed one of six diets containing differing docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) levels. Fish were restricted fed on a pair-fed feeding regime to eliminate variability in feed intake, with two diets fed to satiety to examine the effects of fixed or variable feed rations on EFA requirements. Weight gain, feed intake, feed utilisation, and physical clinical signs were monitored. No effect of dietary DHA and EPA concentration, DHA:EPA ratio or total LC-PUFA level was observed on weight gain, growth rate, feed conversion ratio (FCR), survival or physical clinical health signs (P &gt; 0.05). Satiety fed fish had higher feed intake, final weight, weight gain and growth rate compared to their respective restrictively fed treatments (P &lt; 0.05). No effect of ration level on the responses to DHA concentration was observed. Body fatty acid composition was affected by diet, increasing dietary DHA resulted in higher body tissue DHA concentration, and a similar relationship was observed for EPA. Plasma haemoglobin increased with increasing DHA + EPA levels (P &lt; 0.05) while glutamate dehydrogenase increased for fish fed DHA + EPA in a 1:1 ratio, regardless of total dietary LC-PUFA (P &lt; 0.05). Juvenile barramundi may be fed diets containing as low as 1 g kg− 1 DHA without compromising growth or health status.
Two experiments were conducted to determine the effects of glycerol concentration and Equex STM p... more Two experiments were conducted to determine the effects of glycerol concentration and Equex STM paste on the post-thaw motility and acrosome integrity of epididymal alpaca sperm. In Experiment 1, epididymal sperm were harvested from male alpacas, diluted, and cooled to 4 degrees C in a Lactose cooling extender, and pellet-frozen in a Lactose cryodiluent containing final glycerol concentrations of 2, 3, or 4%. In Experiment 2, epididymal sperm were diluted in Biladyl, cooled to 4 degrees C, stored at that temperature for 18-24 h, and further diluted with Biladyl without or with Equex STM paste (final concentration 1% v:v) before pellet freezing. In Experiment 1, sperm motility was not affected by glycerol concentration immediately (2%: 16.1 +/- 4.6%; 3%: 20.5 +/- 5.9% and 4%: 18.5 +/- 6.6%; P &gt; 0.05) or 3h post thaw (&lt; 5% for all groups; P &gt; 0.05). Post-thaw acrosome integrity was similar for sperm frozen in 2% (83.6 +/- 1.6%), 3% (81.3 +/- 2.0%) and 4% glycerol (84.8 +/- 2.0%; P &gt; 0.05) but was higher 3h post-thaw for sperm frozen in 3% (75.7 +/- 3.8%) and 4% (77.2 +/- 4.1%) than 2% glycerol (66.9 +/- 2.7%; P &lt; 0.05). In Experiment 2, sperm motility was higher immediately after thawing for sperm frozen in the presence of Equex STM (Equex: 21.5 +/- 3.5%; control: 14.4 +/- 2.1%; P &lt; 0.05) but was similar at 3h post-thaw (P &gt; 0.05). Acrosome integrity was similar for sperm frozen with or without Equex STM paste immediately (control: 89.6 +/- 1.2%; Equex: 91.1 +/- 1.4%; P &gt; 0.05) and 3 h post-thaw (control: 69.3 +/- 3.7%; Equex: 59.9 +/- 5.8%; P &gt; 0.05). Sperm cryopreserved in medium containing 3-4% glycerol and 1% Equex STM retained the best motility and acrosome integrity, even after liquid storage for 18-24 h at 4 degrees C prior to cryopreservation.
Artificial insemination (AI) is an important technique in all domestic species to ensure rapid ge... more Artificial insemination (AI) is an important technique in all domestic species to ensure rapid genetic progress. The use of AI has been reported in camelids although insemination trials are rare. This could be because of the difficulties involved in collecting as well as handling the semen due to the gelatinous nature of the seminal plasma. In addition, as all camelids are induced ovulators, the females need to be induced to ovulate before being inseminated. This paper discusses the different methods for collection of camel semen and describes how the semen concentration and morphology are analyzed. It also examines the use of different buffers for liquid storage of fresh and chilled semen, the ideal number of live sperm to inseminate and whether pregnancy rates are improved if the animal is inseminated at the tip of the uterine horn verses in the uterine body. Various methods to induce ovulation in the female camels are also described as well as the timing of insemination in relation to ovulation. Results show that collection of semen is best achieved using an artificial vagina, and the highest pregnancy rates are obtained if a minimum of 150×10(6) live spermatozoa (diluted in Green Buffer, lactose (11%), or I.N.R.A. 96) are inseminated into the body of the uterus 24h after the GnRH injection, given to the female camel to induce ovulation. Deep freezing of camel semen is proving to be a great challenge but the use of various freezing protocols, different diluents and different packaging methods (straws verses pellets) will be discussed. Preliminary results indicate that Green and Clear Buffer for Camel Semen is the best diluent to use for freezing dromedary semen and that freezing in pellets rather than straws result in higher post-thaw motility. Preservation of semen by deep-freezing is very important in camelids as it prevents the need to transport animals between farms and it extends the reproductive life span of the male, therefore further work needs to be carried out to improve the fertility of frozen/thawed camel spermatozoa.
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