Effect of nutritional stress on physiological parameters and seminal attributes of native-crossbred ram in semi-arid tropics.

A prolific three-breed (Malpura, Patanwadi, and Garole) cross Avishaan sheep has been developed in the semi-arid zone to improve farmer's income. Nutritional scarcity is a major limitation in animal husbandry during the dearth period of semi-arid tropics. Therefore, before the inaugural launch of the breed into the field, a study was designed to evaluate the effect of nutritional stress on physiological parameters and seminal attributes of native-crossbred rams in semi-arid tropics. Thus, 16 native adapted (Malpura) and 16 native-crossbred rams were equally distributed into four groups, namely, native control (G1), native nutritional stress (G2), native-crossbred control (G3), and native-crossbred nutritional stress (G4). Both the control groups (G1 and G3) were kept on their maintenance requirement as per their body weight, whereas the nutritional stress groups (G2 and G4) were provided 30% less than their maintenance requirement. The body weight of G4 decline (P<0.05) as compared to their initial weight. The plasma glucose level of G2 and G4 reduced (P<0.05) in comparison with G1 and G3, respectively. The total motile sperm percentage, rapid motile sperm percentage, and sperm viability decrease significantly (P<0.05) within the acceptable limit in native-crossbred rams (G4) under nutritional scarcity. However, the similar blood biochemical along with acceptable seminal attributes of all the rams reflected that native-crossbred rams can cope with the nutritional scarcity in semi-arid tropics and have the potential to contribute to the sustainable small ruminant production system for livelihood security in this region.

Mimicking the cauda epididymal plasma-like osmolality in extender improves liquid preservation of ram semen at 3-5°C.

Cauda epididymis in mammals is known to store mature sperm largely in quiescent state for several weeks without significantly affecting fertility. Hence, the aim of this study was to evaluate the effects of mimicking cauda epididymal plasma (CEP)-like conditions in extender on liquid preservation of ram semen at 3-5°C. Four experiments were conducted in this study: (1) evaluation of physicochemical properties of ram CEP, (2) effect of hyperosmotic solution on sperm motility and functional membrane integrity (FMI), and the effects of (3) CEP-like hyperosmolality (390 vs. 360 mOsmol/kg) and (4) pH in extender (pH 6.5 vs. 6.8) on liquid preservation of ram semen. Sperm treatment with hyperosmotic solution (450 mOsmol/kg) resulted in a decline (P < 0.05) in mass motility (3.5 ± 0.2 vs. 4.3 ± 0.2) and FMI (30.4 ± 3.2 vs. 52.1 ± 2.1%) compared to that with isoosmotic solution (360 mOsmol/kg). Overall, sperm viability, acrosomal integrity, and progressive motility were similar (P > 0.05) while straight-line velocity (77.8 ± 3.1 vs. 71.3 ± 2.7µm/s), linearity (47.4 ± 0.4 vs. 39.5 ± 0.9%), straightness (79.7 ± 0.5 vs. 74.0 ± 0.5%) and beat cross frequency (28.6 ± 0.8 vs. 26.0 ± 0.5 Hz) were higher (P < 0.05) and FMI (65.7 ± 1.5 vs. 75.4 ± 1.1%) was lower (P < 0.05) following liquid-preservation in hyperosmotic extender compared to that in isoosmotic extender. Both total motility (83.3 ± 1.8 vs. 75.4 ± 1.5%) and progressive motility (51.7 ± 2.3 vs. 39.5 ± 1.9%) were higher (P < 0.05) at 48 h of storage in hyperosmotic extender compared to the control. Overall, the seminal attributes were similar (P > 0.05) between the two pH's of the extender. In conclusion, semen extender having CEP-like osmolality but not the pH was superior to extenders having conventional osmolality and pH for liquid preservation of ram semen.Abbreviations: AI: artificial insemination; ALH: amplitude of lateral head displacement; BCF: beat cross frequency; CASA: computer-assisted semen analyzer; CEP: cauda epididymal plasma; ELON: elongation; EYTF: egg yolk-Tris-citrate-fructose; FMI: functional membrane integrity; GLM: general linear model; GPC: glycerophosphatidylcholine; HOS: hypoosmotic swelling; LIN: linearity; pHe: external pH; PROG: progressive motility; S.E.M.: standard error of the mean; SLTF: soya lecithin-Tris-fructose extender; SP: seminal plasma; STR: straightness; VAP: average path velocity; VCL: curvilinear velocity; VSL: straight-line velocity; TM: total motility.

Production of a bioactive recombinant chicken matrix metalloproteinase-11 peptide in Escherichia coli.

Matrix metalloproteinase-11 (MMP-11) is known to be highly expressed in metastatic and most invasive forms of tumors. Being selectively expressed in tumor tissues, MMP-11 is a promising target for immunotherapy against tumors. Here, we report the production of a thioredoxin-tagged bioactive recombinant chicken MMP-11 (cMMP-11) peptide excluding the secretory signal and propeptide in Escherichia coli T7 Express lysY using pET32b(+) vector. High-level expression and purification of the bioactive peptide were achieved by induction with 1.0 mM isopropyl-ß-d-thiogalactopyranoside for 4 H at 37 °C followed by affinity chromatography under denaturing condition and slow dialysis. The recombinant peptide exhibited both caseinolytic and gelatinase activities without requiring activation by 4-aminophenylmercuric acetate. The antisera raised against the peptide in rabbits showed a strong reaction with the whole recombinant peptide as well as 37 kDa cMMP-11 mature peptide and cross-reactivity with a 43 kDa protein in murine breast tumor of 4T1 origin in Western blot analysis. The 43 kDa protein in the tumor homogenate showed immunoreactivity with a monoclonal antibody against human MMP-11, suggesting it to be murine MMP-11 having cross-reactivity with the antisera raised against cMMP-11 peptide. Altogether, the study characterized the production of a bioactive and immunogenic recombinant cMMP-11 peptide in E. coli.