Efeito da lipoproteína de baixa densidade sobre algumas características funcionais dos espermatozóides eqüinos criopreservados

Abstract

Egg yolk is one the main substances used in extenders for cryopreservation of equine semen. Its function is to reduce the cold shock caused by temperature variation during the freezing-thawing process. However, egg yolk contains substances that can have negative effects on cryopreservation and can also carry potentially patogenic substances. Benefits derived from the use of egg yolk are mainly attributed to the presence of low-density lipoproteins (LDL). LDL act either by incorporating their content of phopholipids and cholesterol to the spermatozoa plasma membrane or by their association with seminal plasma proteins, which prevents such proteins to phopholipids and cholesterol from the spermatozoa plasma membrane. Thus, LDL make spermatozoa plasma membrane more stable, reducing their sensitivity to cold shock. This study aimed to investigate the effects of the replacement of the egg yolk in a commercial extender by different concentrations of LDL on some functional characteristics of cryopreserved equine spermatozoa. In experiment 1 (EXP1), the control treatment was the Martin et al. (1979) extender (T0) whereas the LDL treatments had concentrations of 8% (T1), 10% (T2) and 12% (T3). We used semen from five thoroughbred stallions with known fertility presenting at least 70% of sperm motility, sperm vigor of 3 and at most 30% of morphological sperm abnormalities. Semen was collected with a Hannover artificial vagina and immediately separated from its gel portion. After measument of the volume, the semen was centrifugated at 800 g during 5 m with G-DETA extender to eliminate seminal plasma. The pellet was divided in 4 fractions, one for each treatment (T0), (T1), (T2), and (T3). Sperm cells concentration was adjusted to 200 x 106 per ml and place in to 0,5 ml straws. Straws were put in a TK 3000® freezing machine, with cooling at 5ºC, followed by reduction to -120ºC. After wards, straws were immersed in liquid Nitrogen. In Experiment 2 (EXP2), we used semen from 3 Crioulo bred stallions, and the same procedures used in EXP1, although semen centrifugation was done without G-EDTA and sperm concentration was adjusted to 50 x 106 spermatozoa/ml. In EXP 2, (T0) used the Martin et al. extender in the control group (T0) compared against its replacement by 8% LDL (T1). In both EXP1 and EXP2, thawing was done in water-bath at 37°C during 30 s. In EXP1, semen samples were submitted to analysis through the Computer-Assisted Semen Analysis (CASA), with evaluation of: total Motility (MT); speed of trajectory in µ/ms (VAP); curvilinear speed in µ/ms (VCL); and progressive speed in µ/ms (VSL). In EXP2, the characteristics evaluated were MT and response to hypoosmotic swelling test (CHIPO). The dependent variables were submitted to repeated measures analysis of variance, with comparisons of means by LSD test. In EXP1, mean MT% did not differ (P > 0,05). among treatments: T0 (23,3%); T1 (18,0%); T2 (15,1%); and T3 (16,9%). VAP for T0 (86,0µ/ms) was higher (P < 0,05) than for T2 and T3 (77,2 and 77,9µ/ms, respectively), but did not differ (P > 0,05) from T1 (81,5µ/ms). However, VAP was similar for T1, T2 and T3 (P > 0,05). VCL did not differ (P > 0,05), for T0 (158,2µ/ms) and T1 (147,3µ/ms), but it was higher (P<0,05) for T0 than for T2 (138,2/µms) and T3 (141,8/µms). VSL did not differ (P > 0,05) across treatments: T0 (61,3 µ3/ms); T1 (60,6µ/ms); T2 (58,1µ/ms); and T3 (57,8µ/ms). In EXP2, MT in T1 (48,8%) was higher (P < 0,05) than in T0 (35,5%), although no differences (P > 0,05) were observed in the response to CHIPO: T0 (30,1%); and T1 (27,8%). Therefore, LDL at 8% can replace egg yolk in the composition of the Martin et al.(1979) extender for cryopreservation of equine semen, without detrimental effects for semen quality.