- Email: [email protected]
Factors affecting homologous in vitro fertilization assay of boar sperm fertility
Theriogenolog)/ 41:2 49,
1994
FACTORS AFFECTING HOMOLOGOUS IN VITRO FERTILIZATION ASSAY OF BOAR SPERM FERTILITY C. Mat~s, 1 E. Martfnez, 1 J.M. V~zquez/J. Roca, 1 J. Gadea, 1 P. Coy, 2 and S. Ruiz, 2 Departments of ~Anlmal Pathology and 2Animal Biology University of Murcia, 30071 Murcla, Spain It has been hypothesized that homologous In vitro fertilization (hlVF) could be used for the assessment of the fertilizing capacity of boar spermatozoa. However, the actual procedure of hlVF In pigs needs to be simplified and standardized. We have previously shown that Immature pig oocytes can be used In a hlVF assay: these oocytes appear to yield fertilization frequencies comparable to those seen with ovulated oocytes, and large numbers can be obtained from slaughterhouse ovaries (Martinez et al., 1993, Theriogenology 40:547-557). The present study was designed to examine the influence of cumulus cells (Experiment 1), storage time of ovaries from their collection until recovery of oocytes (Experiment 2), and oocyte size (Experiment 3) on penetrability of immature pig oocytes. The hlVF procedure has been previously described (Vdzquez et al., 1993, Mol. Reprod. Develop. 36:84-88). In experiments 1 and 3, the collection of oocytes was completed within 2 hours of slaughter. In experiment 1, cumulus-oocytes complexes (cumulus oocytes) were obtained from slaughterhouse ovaries. About one-half of the complexes were mechanically stripped of cumulus cells (denuded oocytes). Cumulus oocytes and denuded oocytes were exposed to 106 or 107 spermatozoa/ml in a 2:<2 factorial design. Results were analyzed by ANOVA. There was a significant effect of sperm concentration (P<0.001) on oocyte penetrability. A significant (P<0.001) cumulus cells effect also existed on degenerate rates of
Oocytes. Type oocyte Sperm/ml Denuded Cumulus Denuded Cumulus
10x10 e 10x10 e lx10 e lx106
(n)
Degeneration(%) Penetration(%) Sperm/oocyte(X+SEM)
395 349 435 392
42.27a 21.49 b 46.44a 23.72 b
89.03 a 83.94 a 70.38 b 66.55 b
8.11 +0.48 a 8.23-+0.45 a 3.08+-0.53 b 3.94+0.48 b
a,bNumbers within columns with different supercripts differ (P < 0.001). In experiment 2, the oocytes were recovered from ovaries kept in PBS (37°C) for 2, 4 or 6 hours after slaughter of prepuberal gilts. A total 2,838 oocytes was inseminated in four replicates. The storage of ovaries for 2, 4 or 6 hours did not modify the penetration rates (96.09, 96.89 and 97.29, respectively) or the number of spermatozoa per oocyte (13.7, 13.6 and 14.1, respectively) but increased (P<0.05) the degeneration rate of oocytes (18.21, 41.41 and 47.08, respectively). In Experiment 3, after colncubation with spermatozoa, oocytes were washed and adherent spermatozoa and cumulus mass were removed by pipetting. The diameters of fresh oocytes were determined using an ocular micrometer under phase-contrast microscopy (400x). Oocytes were then fixed and stained. Results were analyzed by ANOVA and they are shown in the following table. Size (l~m) <137.5 137.5-145 147.5-155 >155
Diameter(X_+SEM) (n) 127.01 -+0.31a 122 141.27+0.06 b 282 151.11_+0.05 c 371 162.98+0.12 d 254
Degeneration(%) 29.51 30.50 31.54 28.35
Penetration(%) Sperm/oocyte(X_+SEM) 75.58 a 76.75 a 91.34 b 95.60 b
4.83+-0.72 a 15.39+ 1.08 b 19.23+1.05 c 21.91+1.24 c
a'b'C'dNumbers within columns with different supercripts differ (P<0.001). Results of this study indicate that immature pig oocytes with size > 147 i~m obtained from ovaries of 4 hours old after slaugther are adequate for the prediction of the fertilizing ability of boar sperm by hlVF. This research was supported by CICYT nQAGF-92/0521.
C o p y r i g h t © 1994 B u t t e r w o r t h - H e i n e m a n n
249
1994
FACTORS AFFECTING HOMOLOGOUS IN VITRO FERTILIZATION ASSAY OF BOAR SPERM FERTILITY C. Mat~s, 1 E. Martfnez, 1 J.M. V~zquez/J. Roca, 1 J. Gadea, 1 P. Coy, 2 and S. Ruiz, 2 Departments of ~Anlmal Pathology and 2Animal Biology University of Murcia, 30071 Murcla, Spain It has been hypothesized that homologous In vitro fertilization (hlVF) could be used for the assessment of the fertilizing capacity of boar spermatozoa. However, the actual procedure of hlVF In pigs needs to be simplified and standardized. We have previously shown that Immature pig oocytes can be used In a hlVF assay: these oocytes appear to yield fertilization frequencies comparable to those seen with ovulated oocytes, and large numbers can be obtained from slaughterhouse ovaries (Martinez et al., 1993, Theriogenology 40:547-557). The present study was designed to examine the influence of cumulus cells (Experiment 1), storage time of ovaries from their collection until recovery of oocytes (Experiment 2), and oocyte size (Experiment 3) on penetrability of immature pig oocytes. The hlVF procedure has been previously described (Vdzquez et al., 1993, Mol. Reprod. Develop. 36:84-88). In experiments 1 and 3, the collection of oocytes was completed within 2 hours of slaughter. In experiment 1, cumulus-oocytes complexes (cumulus oocytes) were obtained from slaughterhouse ovaries. About one-half of the complexes were mechanically stripped of cumulus cells (denuded oocytes). Cumulus oocytes and denuded oocytes were exposed to 106 or 107 spermatozoa/ml in a 2:<2 factorial design. Results were analyzed by ANOVA. There was a significant effect of sperm concentration (P<0.001) on oocyte penetrability. A significant (P<0.001) cumulus cells effect also existed on degenerate rates of
Oocytes. Type oocyte Sperm/ml Denuded Cumulus Denuded Cumulus
10x10 e 10x10 e lx10 e lx106
(n)
Degeneration(%) Penetration(%) Sperm/oocyte(X+SEM)
395 349 435 392
42.27a 21.49 b 46.44a 23.72 b
89.03 a 83.94 a 70.38 b 66.55 b
8.11 +0.48 a 8.23-+0.45 a 3.08+-0.53 b 3.94+0.48 b
a,bNumbers within columns with different supercripts differ (P < 0.001). In experiment 2, the oocytes were recovered from ovaries kept in PBS (37°C) for 2, 4 or 6 hours after slaughter of prepuberal gilts. A total 2,838 oocytes was inseminated in four replicates. The storage of ovaries for 2, 4 or 6 hours did not modify the penetration rates (96.09, 96.89 and 97.29, respectively) or the number of spermatozoa per oocyte (13.7, 13.6 and 14.1, respectively) but increased (P<0.05) the degeneration rate of oocytes (18.21, 41.41 and 47.08, respectively). In Experiment 3, after colncubation with spermatozoa, oocytes were washed and adherent spermatozoa and cumulus mass were removed by pipetting. The diameters of fresh oocytes were determined using an ocular micrometer under phase-contrast microscopy (400x). Oocytes were then fixed and stained. Results were analyzed by ANOVA and they are shown in the following table. Size (l~m) <137.5 137.5-145 147.5-155 >155
Diameter(X_+SEM) (n) 127.01 -+0.31a 122 141.27+0.06 b 282 151.11_+0.05 c 371 162.98+0.12 d 254
Degeneration(%) 29.51 30.50 31.54 28.35
Penetration(%) Sperm/oocyte(X_+SEM) 75.58 a 76.75 a 91.34 b 95.60 b
4.83+-0.72 a 15.39+ 1.08 b 19.23+1.05 c 21.91+1.24 c
a'b'C'dNumbers within columns with different supercripts differ (P<0.001). Results of this study indicate that immature pig oocytes with size > 147 i~m obtained from ovaries of 4 hours old after slaugther are adequate for the prediction of the fertilizing ability of boar sperm by hlVF. This research was supported by CICYT nQAGF-92/0521.
C o p y r i g h t © 1994 B u t t e r w o r t h - H e i n e m a n n
249