The hypoosmotic swelling test in fresh rabbit spermatozoa.

The hypoosmotic swelling test (HOST) has proved to be a good tool for evaluating the membrane integrity of spermatozoa of various domestic animals including cattle, horses, and swine. However, the best approach for using this technique in rabbit semen has not been tested. The present study aimed to establish the best hypoosmotic solution (HS) for testing membrane integrity in fresh rabbit semen. Sucrose solutions with the following osmolarities were used: 50, 60, 75, 100, 125 and 150mOsm/L. Semen samples (n=30) were collected from five mature White New Zealand rabbits (six collections per rabbit) at 72h intervals. After macroscopic evaluation, 10microL of semen was immediately added to 2mL of each solution and incubated for 1h at 37 degrees C. Sequentially, 20microL of semen diluted in HS were evaluated with oil immersion using a phase-contrast microscope. A total of 200 spermatozoa were counted in at least five different fields, and sperm tails were classified as non-coiled, coiled, and strongly coiled. The respective percentages of spermatozoa with coiled tails (coiled plus strongly coiled) in the six solutions listed above were 54.8, 65.2, 54.3, 53.9, 38.9 and 29.4%. Percentage of strongly coiled spermatozoa was: 40.2, 51.0, 43.2, 41.5, 32.7 and 26.9 for the six solutions, respectively. According to total and strong coiling 60mOsm/L was superior to others treatments (P<0.05). Results suggest that the 60mOsm/L solution would be most desirable for use in HOST in fresh rabbit spermatozoa.

Penetration of zona-free hamster, bovine and equine oocytes by stallion and bull spermatozoa pretreated with equine follicular fluid, dilauroylphosphatidylcholine or calcium ionophore A23187.

Experiments evaluated the ability of follicular fluid (FF), dilauroylphosphatidylcholine (PC12) and the calcium ionophore A23187 (A23187) to induce capacitation in stallion and bull spermatozoa, determined by the ability of the spermatozoa to penetrate zona-free hamster, bovine and equine oocytes. Spermatozoa suspensions were incubated at 37 degrees C in one of the following treatments: 1) a modified Tyrode's medium (BGM3) alone; 2) BGM3 + FF; 3) BGM3 + PC12; 4) BGM3 + FF + PC12; 5) BGM3 + A23187; and 6) BGM3 + FF + A23187. Treated spermatozoa were incubated with zona-free hamster, bovine and equine oocytes for 3 h, after which oocytes were stained to assess spermatozoa penetration. The number of hamster oocytes penetrated by spermatozoa incubated in BGM3 alone (1/30) or in presence of FF (2/31) was significantly lower (P < 0.05) than by spermatozoa treated with PC12 or A23187 (16/30 and 17/30, respectively). Processing stallion spermatozoa either by a swim-up procedure or by centrifugation through a Percoll gradient increased the percentages of motile spermatozoa in the final sample, and spermatozoa collected by both processes penetrated similar numbers of zona-free hamster oocytes (P > 0.05). Although treating spermatozoa with PC12 or A23187 enabled both stallion and bull spermatozoa to penetrate oocytes, higher numbers of bovine oocytes were penetrated by bull spermatozoa (25/30) than by stallion spermatozoa (4/30) regardless of spermatozoal treatment. However, the number of zona-free hamster and equine oocytes penetrated by bull spermatozoa (25/30 and 12/18 respectively) and stallion spermatozoa (17/30 and 15/21 respectively) were similar (P > 0.05). We conclude that both PC12 and A23187 capacitate stallion and bull spermatozoa sufficiently to permit the acrosome reaction to occur, enabling spermatozoa to penetrate homologous and heterologous zona-free oocytes.