Influence of cryoprotectants glycerol and amides, combined with antioxidants on quality of frozen-thawed boar sperm.

The present study was undertaken to examine whether the cooling and freezing extenders containing a mixture of antioxidants (AOs) catalase, Na-pyruvate and mercaptoethanol and one of three types of cryoprotectants (CPs) would be able to improve the quality of frozen-thawed boar sperm. The collected semen, only the sperm-rich fraction, was diluted 1:1 with Androhep plus™ extender, stored at 15°C for 2 h and centrifuged. The centrifuged sperm pellet was re-suspended in lactose-egg yolk extender and divided into four groups for mixing with freezing extenders containing different kinds of CPs at 5°C: (I) glycerol (GLY) as control; (II) GLY with AOs; (III) dimethyl formamide (DMF) with AOs and (IV) dimethyl acetamide (DMA) with AOs. Processed sperm were packaged in 0.25-mL straws and frozen using a controlled rate freezer. After thawing, the diluted thawed sperm were incubated at 38°C for 10 min and was assessed for motility by CASA, membrane/acrosome integrity by FITC-PNA/PI and DNA integrity (DFI) by SCSA. All sperm parameters evaluated, except DFI, were negatively affected (P<0.001) when using DMF (III) or DMA (IV) as CPs instead of GLY (I and II). Total sperm motility was lower (P<0.001) in the samples supplemented with AOs (32.4 ± 1.2, 23.9 ± 1.5, 6.9 ± 0.7, and 10.3 ± 0.9%, for treatments I, II, III and IV, respectively). The quality of sperm frozen in DMF was not different from DMA (P>0.05). There was no difference in DFI among the studied groups (P>0.05). In conclusion, based on the present results, addition of AOs to cooling and freezing extenders and/or replacement of GLY with DMF or DMA could not improve quality of frozen-thawed boar sperm.

New aspects of boar semen freezing strategies.

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2-3% in the freezing media, cooling rates of -30 to -50 degrees C/min, and thawing rates of 1200-1800 degrees C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1x10(9) spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.

Altered mRNA expression patterns in bovine blastocysts after fertilisation in vitro using flow-cytometrically sex-sorted sperm.

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.

Utilization of frozen-thawed epididymal ram semen to preserve genetic diversity in Scrapie susceptible sheep breeds.

The European Union has introduced transmissible spongiform encephalopathy (TSE) resistance breeding programmes for several sheep breeds to cope with the genetic susceptibility to Scrapie infections. Due to the different allele frequencies among breeds, strong selection for ARR alleles is associated with a loss of genetic diversity in small populations and in larger populations with unfavourable ARR allele frequencies. To ensure maintenance of genetic diversity, an adhoc cryopreservation programme was initiated employing epididymal sperm from 109 rams representing 16 different breeds within one breeding season. Epididymal semen was chosen for this adhoc programme because time consuming training of rams for ejaculated semen collection via an artificial vagina was not possible. Prior to freezing, average sperm motility was 79.7% and acrosome integrity was 93.7%. After freezing, these levels were decreased to 60.5 and 72.8%, respectively. An insemination trial using frozen-thawed epididymal semen resulted in a lambing rate of 87.5%. Results show that this semen preservation method is robust and efficient and associated with high fertility. It may also be useful for other animal species.