Fernando Andrade Souza

Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm

Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in liquid nitrogen; (T2) T1 with the addition of cholesterol before cooling (the cholesterol was incorporated to the sperm membranes with the methyl-beta-cyclodextrin-cholesterol complex); (T3) T2 with post-thaw removal of the cholesterol with 0.052 mg methyl-beta-cyclodextrin/50 x 10(6) sperm; and (T4) T3 with 0.156 mg methyl-beta-cyclodextrin/50 x 10(6) sperm. Sperm progressive motility and functional integrity of sperm plasma membranes were evaluated microscopically and by the hyposmotic swelling test, respectively. Using flow cytometry, physical integrity of sperm plasma membranes was assessed with propidium iodide, acrosomal integrity with fluoresceinated lectin peanut agglutinin, and rate of sperm acrosome reaction induced with of the calcium ionophore A23187. Cholesterol inclusion (T2) increased the proportion of frozen-thawed sperm with intact plasma membrane. Nevertheless, sperm from T2 (9.3+/-5.9%) had a lower rate of acrosome reaction after induction, compared to the control group (16.5+/-11.0%). After cholesterol removal, there was no increase in the induced acrosome reaction rate (T3: 11.3+/-7.1% and T4: 11.8+/-9.9%). Perhaps the cyclodextrin concentrations used were too low to remove sufficient cholesterol from sperm membranes to restore the ability of cryopreserved sperm to undergo an acrosome reaction. Regardless, the addition of cholesterol to improve post-thaw sperm integrity, and its subsequent removal, still has potential for cryopreservation of stallion sperm.

Leptin and insulin in the seminal plasma of zebu bulls in peripuberty

In order to determine the profile of leptin and insulin values in seminal plasma and their correlations with sperm patterns during the peripuberty, a total of 16 crossbred Gyr dairy bulls were monitored from 60 days before to 60 days after puberty, separated into two groups, early and regular, according to the onset of puberty. Hormone values were determined by radioimmunoassay after gel filtration in fast performance liquid chromatography and semen patterns, according to the Brazilian College of Animal Reproduction. Insulin concentrations in seminal plasma did not differ between groups, nor between the ages, with the pooling of the data showing average of 35.15±16.93, 36.60±26.21, 26.82±09.81, and 43.56±31.71 32.24±16.71 U/mL for the pubertal period of -60, -30, 0, +30 and +60 days, respectively. These values were not correlated with sperm parameters, but differed in the group of animals with the highest percentage of major defects (31.08±18.58 U/mL) compared with those with lower percentage of these sperm defects (40.01±25.37 U/mL). Leptin was not correlated with sperm parameters and did not differ between groups or stage of sexual development, with concentrations of 23.10±10.43, 24.35±9.42, 22.41±9.90, 23.76±9.69 and 24.99±11.42 ng/mL for the periods of puberty aforementioned, respectively, after being grouped. These results demonstrated the binding of insulin to the structural quality of the sperm cell, and this hormone is indicated in andrologycal evaluation of future breeding soundness.