K. M. Morton

Cryopreservation of epididymal alpaca (Vicugna pacos) sperm: a comparison of citrate-, Tris- and lactose-based diluents and pellets and straws

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 < 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 < 0.05), although by 3 h post-thaw motility was similar for pellets and straws (P > 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.

Effect of glycerol concentration, Equex STM® supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen-thawed epididymal alpaca (Vicugna pacos) sperm

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 > 0.05) or 3h post thaw (< 5% for all groups; P > 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 > 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 < 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 < 0.05) but was similar at 3h post-thaw (P > 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 > 0.05) and 3 h post-thaw (control: 69.3 +/- 3.7%; Equex: 59.9 +/- 5.8%; P > 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.

Effect of Aspiration Pressure during Oocyte Harvesting on Oocyte Recovery and in vitro Development of Ovine Oocytes

Oocytes from abattoir-sourced ovine ovaries were aspirated from 2- to 4-mm follicles using 25, 50 or 100 mmHg pressure and an aspiration pump, or a needle (20-G) and syringe (2.5 ml) and subjected to in vitro maturation, fertilization and culture to determine the effect of aspiration pressure on the number and quality of oocytes recovered, and early embryonic development. Oocyte recovery rate was similar between groups (range: 57.1-73.1%; p > 0.05). The number and percentage of grade I and II oocytes recovered was reduced for 100 mm (24.5 +/- 3.6 and 31.1 +/- 6.1%) compared with 25 mm (51.4 +/- 7.0 and 60.2 +/- 7.8%) and 50 mm pressure (40.8 +/- 5.6 and 50.3 +/- 4.4%) and a syringe (40.3 +/- 12.0 and 45.2 +/- 2.1%; p < 0.05). Oocyte cleavage was similar for all groups at 24 (range: 30.9-49.6%) and 48 h post-insemination (49.7-65.5%), but blastocyst formation (% cleaved oocytes) was lower for oocytes aspirated with 25 mm (37.8%) than 50 (69.2%) or 100 mm (67.2%) pressure, and a syringe (72.0%; p < 0.05). Embryo production efficiency (% of oocytes cultured developing to the blastocyst stage) was higher for oocytes aspirated with 50 mm (45.4%) and 100 mm pressure (43.8%) and a syringe (45.0%) than 25 mm pressure (18.8%; p < 0.05). These results demonstrate that the aspiration of ovine oocytes with an aspiration pressure of 50 mm, or aspiration with a needle and syringe are equally efficacious for the in vitro production of embryos.

Effects of lamb age, hormone stimulation and response to hormone stimulation on the yield and in vitro developmental competence of prepubertal lamb oocytes

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 3-4-week-old lambs only (P < 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 > 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 < 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.

Quality Parameters for Alpaca (Vicugna pacos) Semen are Affected by Semen Collection Procedure

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.

An efficient method of ovarian stimulation and in vitro embryo production from prepubertal lambs

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 was related to both bodyweight and growth rate (P < 0.05), although ovarian weight and the number of ovarian follicles were not related to either bodyweight or growth rate. Oocyte cleavage varied between hormone-stimulated lambs (0.0-93.0%; P < 0.05), and 484/775 (62.2%) of the oocytes cultured cleaved. Oocytes from 17 of the 19 lambs (89.5%) developed to the blastocyst stage in vitro , and the proportion of zygotes forming a blastocyst (by Day 7) ranged from 0.0 to 66.7% for individual lambs. Overall, 33.9% of zygotes (n = 164) developed to the blastocyst stage, producing 8.6 +/- 2.8 blastocysts per lamb.

Quantification of the DNA Difference, and Separation of X- and Y-Bearing Sperm in Alpacas (Vicugna pacos)

Sperm sexing is an emerging reproductive technology which has been successfully used to produce offspring of a pre-determined sex in domestic and wildlife species but has yet to be applied to New World camelids. The aims of the present study were to (i) optimize the Hoescht 33342 (H33342) staining concentration for the flow cytometric separation of X and Y chromosome-bearing alpaca (Vicugna pacos) sperm nuclei, (ii) separate alpaca sperm nuclei into high purity (>90%) populations bearing the X- and Y-chromosome and (iii) determine the DNA difference between X- and Y-bearing sperm in alpacas. Semen was collected from alpacas and sperm nuclei stained with H33342, incubated and analysed using a high-speed cell sorter (SX-MoFlo). H33342 staining concentrations of 36, 54, 72 or 90 microm did not affect the proportion of correctly oriented sperm nuclei (43.3 +/- 3.9, 46.4 +/- 3.7, 44.5 +/- 4.0 and 51.1 +/- 2.5% respectively) nor the speed of sorting (1381 +/- 160, 1386 +/- 123, 1371 +/- 133 and 1379 +/- 127 sperm nuclei/s). Sort reanalysis determined high levels of purity for X- and Y-enriched populations (96.6 +/- 0.7% and 96.1 +/- 1.1% respectively). The DNA difference, based on fluorescence intensity (determined by the SX-MoFlo), was 3.8 +/- 0.06%. These data demonstrate for the first time that alpaca sperm nuclei can be separated into high purity populations and the potential for applying sperm sexing technology to New World camelids.