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The objective of this study was to assess the utility of the magnetic-activated cell sorting (MACS) technique used for improving characteristics and quality of insemination doses by the elimination ofapoptotic rabbit spermatozoa from a heterospermic pool (Experiment 1) as well as from the ejaculates of individual bucks (Experiment 2). Superparamagnetic microbeads conjugated with annexin V eliminated spermatozoa with externalized phosphatidylserine via MACS. The control (untreated) and magnetically separated spermatozoa (in both E1 and E2) were used for artificial insemination of hormonally treated rabbit does. MACS separation of spermatozoa yielded two fractions: annexin V-negative (AnV) and annexin V-positive (AnV+). The CASA analysis after MACS sperm sorting revealed that the proportion of apoptotic spermatozoa in the semen of New Zealand White bucks varied from 7 to 20%. Transmission electron microscopy revealed that MACS treatment might eliminate spermatozoa with membrane damages and released acrosomal matter. However, the MACS separation (in both E1 and E2) did not affect the reproductive parameters of rabbit does.

The aim of this study was to verify whether the separation and elimination of the apoptotic fraction in rabbit semen using a MACS technique may improve sperm fertility potential and consequently rabbit kindling rate. Semen samples from 25 New Zealand White (NZW) rabbit males were collected using an artificial vagina and evaluated using the CASA system for concentration and motility. For artificial insemination the best 11 bucks were chosen based on motility parameters. Their ejaculates were mixed to make a heterospermic pool and routinely diluted in a commercial insemination diluent (MiniTüb, Tiefenbach, Germany) at a ratio of 1:6. Diluted heterospermic spermatozoa were filtered through a Sartorius filter to wash out seminal plasma, re-diluted in binding buffer (Annexin V Microbead Kit, Miltenyi Biotec, Germany) at a ratio of 1:3.66 and divided into two groups: an experimental group intended for MACS separation and control group without MACS separation. Then hormonally treated females of NZW rabbits were inseminated with fresh doses of filtered heterospermic semen (n = 27; 0.5 ml I.D. per female) and MACS separated semen (n=28; 0.5 ml I.D. per female). Separation and subsequent elimination of apoptotic spermatozoa (positive selection) from the insemination dose (after negative MACS selection) was verified under in vivo conditions on the basis of increased kindling rate in the experimental group in comparison with kindling rate in the control group (81.3% vs. 73.8%). In conclusion, elimination of apoptotic spermatozoa by the use of the MACS technique results in a slight improvement in kindling rate of rabbit does.

Summary: Rabbits are an important animal species for meeting the nutritional requirements of the world's growing population due to the high conversion rate of feed. In most countries, the rabbit industry currently relies on artificial insemination with fresh or chilled and frozen–thawed spermatozoa. Various factors during the freezing process, including diluents, sperm preparation and freezing techniques, antioxidants, sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for the poor sperm quality post thaw. Despite the extensive progress reached in the field of rabbit sperm cryopreservation, new methodological approaches that could overcome problems in sperm cryopreservation are necessary. The aim of this review was to describe the factors that affect the cryopreservation of rabbit sperm. [ABSTRACT FROM AUTHOR]