A. Donovan

Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm.

Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 degrees C and 15 degrees C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 degrees C maintained acceptable motility and viability up to 72h compared with that of storage at 15 degrees C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250muL semen at a sperm concentration of 20x10(6)/mL. Semen was stored at 5 degrees C and tested after 6, 24, 48, and 72h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 degrees C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72h after semen collection. Cleavage rate was recorded at 48h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24h resulted in higher rates than those for storage at 72h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 degrees C, and used to inseminate ewes on the day of collection or at 24, 48, and 72h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly (P<0.001) up to 72h after semen collection.

Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen-thawed semen

Several procedures have been proposed to assess structural and functional characteristics of cryopreserved ram semen but none so far have yielded consistent relationships with in vivo fertility. The objectives of this study were to evaluate several sperm function tests as potential markers of in vivo ram fertility (determined by pregnancy rate in ewes) using frozen-thawed semen. In experiment 1, frozen-thawed straws (n=3 per ram) of semen from three high and three low fertility rams were assessed using fluorescent microscopy for (1) progressive motility, (2) viability and, (3) acrosomal status. In experiment 2, frozen-thawed straws (n=3 per ram) of semen from 18 rams of known fertility were analysed using either computer-assisted sperm analysis (CASA) for eight motion characteristics or flow cytometric staining for: (1) viability and acrosomal status, (2) plasma membrane status and capacitation-like changes, and (3) live cells following an osmotic resistance test (ORT). In experiment 3, platelet-activating factor (PAF) was isolated from straws (n=2 per ram) of semen using high-pressure liquid chromatography (HPLC) and quantified using HPLC-tandem mass spectrometry for 18 rams. In experiment 1, no association was found between motility, viability (% live) or acrosomal status (% damaged, % intact and % reacted) and in vivo fertility. In experiment 2, no correlation was found between motility (CASA), viability (% live), acrosomal status (% live, % live intact and % reacted), capacitation status (% capacitated, % non-capacitated), plasma membrane stability (% dead) and % live cells following ORT and ram in vivo fertility. In experiment 3, there was no relationship between PAF content in spermatozoa and ram fertility. In conclusion, we were unable to relate the in vivo fertility of rams with in vitro functional tests of their frozen-thawed semen and suggest that the fertility of a given semen sample cannot easily be quantified using available in vitro tests.

Ewe breed differences in fertility after cervical AI with frozen-thawed semen and associated differences in sperm penetration and physicochemical properties of cervical mucus.

The objective of this study was to examine sperm penetration through cervical mucus and associated physicochemical properties of cervical mucus from Belclare and Suffolk ewes - two breeds with divergent pregnancy rate following cervical AI using frozen-thawed semen. In Experiment 1, sperm penetration through cervical mucus was assessed in 15 Belclare and 15 Suffolk ewes at 30, 48 and 57h post sponge removal. In Experiment 2, rheological properties of mucus from 17 Belclare and 19 Suffolk ewes at 48 and 57h post sponge removal were determined. In Experiment 3, 20 Belclare and 20 Suffolk ewes were used to assess mucus ferning and pH collected at 42, 48, 57 and 65h post sponge removal. In Experiment 1, a higher number of sperm penetrated cervical mucus from Belclare ewes at 48h, reflected by a breed by time interaction (P=0.05). In Experiment 2, mucus from Suffolk ewes tended to have higher elastic and complex moduli than that from Belclare ewes (P=0.06) regardless of time of collection. There was no effect of ewe breed on the viscous modulus. In Experiment 3, there was a significant effect of time post sponge removal on ferning (P<0.01), but there was no effect of breed. There was no effect of time or breed on mucus pH. It is concluded that breed differences in the rheological properties of cervical mucus affect the ability of sperm to swim through cervical mucus and this may explain breed differences in fertility observed after cervical AI using frozen-thawed semen.

Effect of site of deposition on the fertility of sheep inseminated with frozen-thawed semen

The widespread use of artificial insemination (AI) in sheep is currently prevented due to the lack of a cost effective insemination technique utilising frozen-thawed semen. The objective of the present study was to determine if the deposition of frozen-thawed semen in the vaginal fornix would result in a pregnancy rate comparable to that achieved following cervical insemination. Multiparous ewes of various breeds were synchronised and inseminated into either the vaginal fornix (n=78) or the cervix (n=79), at 57 h post sponge removal, with frozen-thawed semen. Information on mucus secretion and the depth to which it was possible to penetrate the cervix at insemination (cervically inseminated ewes only) was recorded at the time of AI. Pregnancy rate was subsequently determined either by return to service (oestrus) or after slaughter 30 days post insemination. Insemination site did not significantly influence pregnancy rate using frozen-thawed semen (36.2% compared to 27.6% for cervical and vaginal fornix insemination, respectively; P=0.26). Whilst depth of cervical penetration was positively associated with pregnancy rate (P<0.05), this association needs to be interpreted with caution as none of the ewes where the cervix could not be penetrated (score=0) was pregnant. In conclusion, pregnancy rate following insemination of frozen-thawed semen into the vaginal fornix was within 10% points of that obtained following cervical AI of frozen-thawed semen. As insemination into the vaginal fornix is technically easier than cervical insemination, it may be more practical for use in large scale applications.