Mammalian spermatozoa remain immotile and metabolically inactive in the cauda epididymidis, thus maintaining fertility for several weeks. The aim of this study was to functionally characterise and evaluate the effect of cauda epididymal plasma (CEP) on liquid preservation of ram spermatozoa. Four experiments were conducted to investigate the effects of: (1) CEP and its fractions on sperm motility; (2) CEP (10%, 15%, 20% v/v) on liquid preservation of ram spermatozoa; (3) seminal plasma (SP; 20%, 30%, 50% v/v) on liquid-preserved spermatozoa; and (4) both CEP and post-storage SP treatment on sperm characteristics. Biochemical characterisation of ram CEP revealed high protein (30.9 mg mL−1), catalase (68.9 IU mL−1), alkaline phosphatase (17.5 IU mL−1) activities and total antioxidant capacity (1112 µM Trolox equivalent). Progressive motility of prewashed cauda spermatozoa was reduced (P < 0.05) by CEP or its protein-rich fraction compared with protein-free plasma or phosphate-buffered saline. After 48 h storage, total motility, rapid motility (average path velocity >75 µm s−1; 53.9%, 73.5% and 71.8% with 0, 15% and 20% CEP respectively) and straight line velocity (86.3, 102.1 and 102.4 µm s−1 with 0, 15% and 20% CEP respectively) were significantly (P < 0.05) higher in the CEP-treated groups than the control. Viability and acrosomal integrity were similar between groups; however, functional membrane integrity was higher (P < 0.05) in the 15% CEP-treated group. Treatment of liquid-preserved spermatozoa with either 20%, 30% or 50% SP improved (P < 0.05) rapid motility and kinematics at each time point of storage compared with control. In conclusion, liquid preservation of ram spermatozoa in the presence of 15% or 20% CEP and post-storage treatment with SP significantly improve sperm characteristics.

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The GMM sheep is a carrier of Booroola fecundity (FecB) gene, which produces the twins and triplets in one lambing. The homozygous carrier GMM (FecBBB), non‐carrier GMM and Malpura (FecB++) ewes were synchronized by progesterone sponges, and the plasma progesterone concentration was measured by RIA. The results showed that the progesterone concentration did not differ significantly (p > .05) in homozygous carrier GMM (5.74 ± 1.2 ng/ml), non‐carrier GMM (5.42 ± 1.4 ng/ml) and non‐carrier Malpura ewes (5.67 ± 1.5 ng/ml). Further, quantitative expression of BMP factors/receptors and SMAD signalling genes were analysed in the ovaries of sheep by qRT‐PCR. The study showed that the expression of BMP2 was slightly higher (p > .05) in carrier GMM than that of non‐carrier GMM, but it was almost similar to Malpura ewes. Expression of BMP4 and BMP7 was significantly higher (p < .001; p < .05) in carrier GMM than that of non‐carrier GMM and Malpura ewes. Although BMP6 expression was higher (p > .05) in carrier GMM than that of non‐carrier GMM, but lower (p > .05) than the Malpura ewes. Expression of BMP15 (p < .05), GDF5 (p < .01) and GDF9 (p < .05) was significantly higher in carrier GMM than non‐carrier GMM ewes. Surprisingly, BMPR1B expression was significantly higher (p < .001) in non‐carrier GMM and Malpura than the carrier GMM ewes, while TGFβRI did not differ significantly (p > .05) among both GMM genotypes. On the other hand, expression of BMPR1A (p > .05) and BMPRII (p < .05) was higher in carrier GMM than the non‐carrier GMM, but significantly lower (p < .001) than the Malpura ewes. It was interesting to note that the expression of SMAD1 (p > .05), SMAD2 (p < .001), SMAD3 (p < .05), SMAD4 (p < .001), SMAD5 (p < .001) and SMAD8 (p < .001) was lower in the carrier GMM than that of non‐carrier GMM ewes. It is concluded that the FecB mutation alters the expression of BMPR1B and SMAD signalling genes in the ovaries of homozygous carrier GMM ewes.

The present study was conducted to analyze the mRNA expression of the BMP/SMAD signaling and steroidogenesis associated genes in the granulosa cells (GCs) of newly developed Booroola homozygous carrier GMM (FecB BB ) and non-carrier GMM (FecB ++ ) ewes through qRT-PCR. Results showed that the expression of BMP2 (P < 0.05) and BMP6 (P < 0.01) was significantly higher in the GCs of the homozygous carrier GMM (FecB BB ) than the non-carrier GMM (FecB ++ ) ewes, while the expression of BMP4 was significantly higher (P < 0.001) in the GCs of non-carrier GMM (FecB ++ ) than the homozygous carrier GMM (FecB BB ). In comparison, the expression of TGFβR1, BMPR1A, BMPR1B, and BMPRII was not significantly different between GCs of the homozygous carrier GMM (FecB BB ) and non-carrier GMM (FecB ++ ) ewes. Similarly, the expression of SMAD1, SMAD2, SMAD3, SMAD4, and SMAD5 was not significantly different between GCs of homozygous carrier GMM (FecB BB ) and non-carrier GMM (FecB ++ ). Further, expression of the INHIBIN, P4R, CYP11A1, and 3βHSD1 genes were not significantly different among the GCs of homozygous carrier GMM (FecB BB ) and non-carrier GMM (FecB ++ ), while the expression of StAR was significantly higher (P < 0.01) in the GCs of homozygous carrier GMM (FecB BB ) than that of GCs of non-carrier GMM (FecB ++ ) ewes. It is concluded that the FecB mutation significantly up-regulates the expression of BMP2, BMP6, and StAR genes and down-regulate the expression of BMP2 in granulosa cells of newly developed GMM ewes.