Effect of L-cysteine Supplementation on Cryopreservation of Black Bengal Buck Semen

Background: Black Bengal goat is one among the valuable goat breeds of India. Semen preservation and artificial insemination are practical technologies for conservation and improving livestock germplasm. Various biochemical changes that occur during cryopreservation of semen, results in loss of sperm cell structure and their functions. Inclusion of antioxidants to the semen extender has been reported to have positive effects on sperm recovery in various livestock species. The present experiment was carried out to evaluate the effect of antioxidant L-cysteine on post freeze thaw sperm characters, lipid peroxide level and superoxide dismutase (SOD) activity during cryo­preservation of Black Bengal buck semen. Methods: Twenty numbers of semen ejaculates were collected from Black Bengal bucks, antioxidant L cysteine was added to the semen extender @ 0, 1 and 2 mM in control group (CC), treatment group 1 (CT1) and treatment group 2 (CT2), respectively. Semen samples were frozen in liquid nitrogen and measured post freeze-thaw in vitro sperm characters, malondialdehyde (MDA) concentration and SOD activity. Result: Though there was no significant improvement in post thaw sperm motility, functional membrane integrity and viability count, supplementation of L-cysteine significantly improved the percentage of sperm cells with intact acrosome and significantly (P less than 0.05) reduced the level of malondialdehyde. Post thaw SOD activity was found significantly lower in the antioxidant treated groups than the control. It is concluded that supplementation of L cysteine is effective in controlling lipid peroxidation and conserving in vitro sperm characters during freezing of Black Bengal buck semen.

Effect of Butylated Hydroxytoluene and Tocopherol Supplementation on In vitro Sperm Characters during Cryopreservation of Black Bengal Buck Semen

Background: Black Bengal goat is one of the important goat breeds of India. Cryopreservation of semen and artificial insemination are effective techniques for improving goat breeding programs. Various biochemical and functional changes that occur during freezing-thawing process results in poor post thaw sperm recovery. Supplementation of antioxidants to the extender has been reported to have positive effects on semen cryopreservation in various species. The present study was undertaken to assess the effect of antioxidants butylated hydroxyl toluene (BHT), α tocopherol on post thaw in vitro sperm characters, lipid peroxide level and superoxide dismutase (SOD) activity during cryo­preservation of Black Bengal buck semen. Methods: Semen was collected from bucks by artificial vagina, antioxidant BHT was added to the Tris egg yolk extender @ 0, 1 and 2 mM/ml in control group (BHTC), treatment group 1 (BHTT1) and treatment group 2 (BHTT2), respectively. Similarly, tocopherol was added @ 0, 1 and 2 mg/ml in control group (TFC), in treatment group 1 (TFT1) and treatment group 2 (TFT2), respectively. Each antioxidant was tested with 20 semen ejaculates. Semen samples were frozen in liquid nitrogen and post freeze-thaw in vitro sperm characters, malondialdehyde (MDA) concentration and SOD activity were measured. Result: Post thaw sperm motility, functional membrane integrity and viable count were significantly (P less than 0.05) higher in the BHT and α tocopherol supplemented groups than control groups. Significantly (P less than 0.05) higher acrosome intact cells were recovered in BHTT1, BHTT2 and TFT2 groups than the control and TFT1 groups. Further, BHT supplemented groups had significantly lower level of MDA than the control, while the supplementation of α tocopherol could not control the generation of MDA. Post thaw SOD activity was found significantly lower in the antioxidant treated groups than their respective controls. It is concluded that supplementation of BHT and α tocopherol were found to be more promising in conserving in vitro sperm characters from cryo-damages during freezing of Black Bengal bucks semen.

The sperm longevity and freezability in the modified BHSV extender of Thai Pradu-hangdum chicken

Pradu-hangdum is a distinctive Thai native pure breed chicken. To conserve the pure breed of this chicken species, the artificial insemination was invested. The key to the highly successful output of this technique is high quality of semen. Hence, the objective of the present study is to characterize the Pradu-hangdam sperms and their longevity and freezability in the BHSV extender. The semen of the 30 randomized male chicken were collected. The macroscopic and microscopic examination were used to determine the sperm characters. The results revealed that the Pradu-hangdam sperms contained normal a spiral-shaped head, mid-piece and tail and normal white cream color. The mean number of sperm concentration was 5.24×109 ± 1.54 sperms/mL. The mean volume was 0.22 ± 0.08 mL. The results of longevity in mean total motility of sperm was 85.20%, 56.00% and 36.33% storage for 1, 24, and 48 hours after semen collection in extender, respectively. The longevity of sperm storage in extender decreased significantly at 24 and 48 hours (P<0.05). The freezability of Pradu-hangdam semen significant decreased from 81.45% to 57.02% of motility (P<0.05) but in the range of acceptable result for insemination. In conclusion, this study provides the basic knowledge of sperm characters and their longevity which decreases in relation to the time after collection even though it was preserved and frozen in the BHSV of the acceptable data. Furthermore, the freezing technique and fertility rate should be a further study in the long-term preservation of Pradu-hangdam sperms.

Sperm ultrastructure in the ocean quahog Arctica islandica (Arcticidae) and Neotrapezium sublaevigatum (Trapezidae), with a discussion of relationships within the Arcticoidea and with other Euheterodonta (Bivalvia)

ABSTRACT Sperm ultrastructure is described for the ocean quahog Arctica islandica (Linnaeus, 1767) (Arcticidae), a long-lived, and commercially and phylogenetically important marine bivalve from the North Atlantic, and for Neotrapezium sublaevigatum (Lamarck, 1819), an Indo-Pacific member of the only other family of Arcticoidea (Trapezidae). Spermatozoa of A. islandica consist of (in anterior to posterior sequence): an elongate-conical, deeply invaginated, acrosomal vesicle (length 2.0 ± 0.2 μm; invagination occupied by a granular subacrosomal material); a straight, anteriorly-tapered, rod-shaped nucleus (length 6.6 ± 0.4 μm); a short (approximately 0.8 μm) midpiece consisting of two orthogonally arranged centrioles, surrounded by four (approximately 75% of spermatozoa observed) or, less commonly, five (approximately 25% of spermatozoa observed) spherical mitochondria; nine satellite fibres connecting the distal centriole to mitochondria and the plasma membrane; and a flagellum (length 60 ± 5.0 μm, with 9+2 axoneme), originating from the distal centriole. Contents of the acrosomal vesicle of A. islandica are differentiated into a very electron-dense basal ring (with reticulate structure) and two less electron-dense zones. Spermatozoa of N. laevigatum (Lamarck, 1819) differ substantially from those of A. islandica and are characterized by: a rounded-conical, deeply invaginated, acrosomal vesicle (length 0.43 ± 0.2 μm), with a curved basal ring and two less conspicuous components; a barrel-shaped nucleus (length 1.6 ± 0.5 μm) with a broad apical depression accommodating the base of the acrosomal vesicle; a midpiece composed of five (approximately 80% of spermatozoa observed) or four (approximately 20% of spermatozoa observed) mitochondria. Centriolar and flagellar details are essentially as for A. islandica, and putative glycogen deposits are associated with the distal centriole and mitochondria in both species. Sperm data corroborate recent transcriptomic analyses separating Arcticidae and Trapezidae in different imparidentian clades. Based on sperm morphology, A. islandica would appear more closely related to the Glauconomidae of the Cyrenoidea than to the Trapezidae, Veneroidea or any other previously examined group of euheterodonts, suggesting that it could be the only living member of the Arcticoidea. The relationships of the Trapezidae remain uncertain, with apparent sperm similarities to members of several groups of euheterodonts (e.g. Tellinoidea, Pholadoidea, Galeommatoidea), while several potentially closely related key taxa (e.g. Glossidae) remain unstudied for sperm characters.

Heparin Binding Proteins of Black Bengal Buck Semen and Their Correlation with Sperm Characters and Freezability

This experiment was conducted to study the electrophoretic characters of heparin binding proteins (HBP) of Black Bengal buck semen and their correlation with sperm characters and cryo-survivability. Semen ejaculates (n=20/buck) were collected from nine bucks and in vitro sperm characters were evaluated at collection, after equilibration and after freeze - thawing. HBP were isolated through heparin column and discontinuous Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed to assess molecular weight. Significant difference (plessthan0.01) were observed among the bucks in sperm characters and freezability. Eight protein bands of 17 to 180 kDa in seminal plasma and 7 bands in sperm were found. 180 -136 kDa HBP of seminal plasma and 134-101 kDa HBP of sperm had showed high correlation with in vitro sperm characters. Further studies on identification of these proteins and their correlation with in vivo pregnancy are needed to find their role as marker for buck selection.

Sperm ultrastructure in Culmenella rezvoji (Lindholm, 1929) (Gastropoda: Hygrophila)

The taxonomic position of the genus within Hygrophila remains uncertain. The spermatozoa of , a species from the Far East of Russia, were examined using a combination of light, scanning and transmission electron microscopy with the objective to assess the utility of sperm characters for clarifying the phylogenetic relationships of the genus. The spermatozoa of C. rezvoji are divided into four regions: head, midpiece, glycogen piece and endpiece. The head contains a slender, cone-shaped acrosome and a conical nucleus with a sinistrally coiled keel. The acrosome consists of an apical vesicle and a thick-walled pedestal with an electron lucent canal partially filled with a patchy electron-dense material. The midpiece contains the mitochondrial derivative that encloses apically three parallel glycogen-filled tracts (helices) positioned in such a way that in the sperm cross section two helices lie opposite each other and equidistant from the third helix. The surface of the sperm above one of the helices forms a high, narrow ridge; the ridges above the remaining two helices have a much lower profile. The boundary between the midpiece and glycogen piece is demarcated by a constriction (annulus) consisting of an anterior electron-dense ring and a posterior conical cylinder connected to the ring with thin filaments. The structure of spermatozoa in Culmenella is consistent with the general pattern of sperm morphology common to all studied species of Hygrophila, but the spermatozoa of Culmenella also have distinctive characters (three glycogen helices and high-profile surface ridge in the apical portion of the midpiece) that should be potentially useful in resolving the taxonomic position of this genus.

Electrophoretic profile of seminal proteins and their correlation with in vitro sperm characters in Black Bengal buck semen

Aim: This study aimed to study the electrophoretic properties of seminal plasma and sperm proteins of Black Bengal buck semen and their correlation with in vitro sperm characters and freezability. Materials and Methods: Semen ejaculates from nine Black Bengal bucks were collected by artificial vagina (n=20/buck). Ejaculates were evaluated for in vitro sperm characters and electrophoretic profile of seminal protein. In vitro sperm characters were evaluated immediately after collection, after completion of equilibration period, and after freeze-thawing. For seminal protein studies, seminal plasma proteins were precipitated by ice-cold ethanol method, and sperm proteins were extracted by Triton X detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to assess the molecular weight of seminal proteins. Correlation between in vitro sperm characters and protein bands was determined by Pearson's correlation coefficient, and two-way ANOVA was applied to find the individual buck differences. Results: Significant difference (p<0.01) among the bucks was noticed in the in vitro sperm characters evaluated at all the three stages of semen evaluation such as immediately after collection, after completion of equilibration period, and post-freeze thawing. Progressive loss of sperm motility, membrane integrity, and other in vitro sperm characters were noticed during cryopreservation. A total of ten protein bands in the molecular weight ranging from 17 to 180 kDa were found in the SDS-PAGE of seminal plasma proteins, while nine bands of 17-134 kDa were observed in sperm proteins. Seminal plasma proteins of molecular weight 75, 62-49, 20, and 17 kDa and sperm proteins of 75, 20, and 17 kDa were present in all the nine bucks (100%) screened, and variation among the bucks was noticed for the presence of other proteins. Seminal plasma protein of 180-134 kDa showed a negative correlation with individual motility (−0.716) and functional membrane integrity of sperm cells (−0.724) in post-freeze-thaw analysis and 48 kDa protein had a positive correlation with individual motility (0.649) and functional membrane integrity of sperm cells (0.664) in post-thaw analysis. Sperm proteins of 63 kDa had a negative correlation (−0.616) with sperm concentration in neat semen. Conclusion: Variation among the bucks was noticed in the in vitro sperm characters and semen freezability. Correlation between seminal proteins and in vitro sperm characters and semen freezability had been found which might be useful as a tool to select breeding bucks.