L-carnitine is a survival factor for chilled storage of rooster semen for a long time

Rooster sperm is sensitive to cooling, which restricts procedures to store sperms for extended periods of time for artificial insemination of commercial flocks. This study was conducted to evaluate the suitability of adding L-carnitine (LC) to chilled-storage of rooster sperm and its effects on sperm quality parameters and its fertility potential during storage at 5 °C. Pooled semen from roosters were divided into six equal aliquots and diluted with media supplemented with different concentrations of LC (0, 0.5, 1, 2, 4 and 8 mM LC). Diluted semen samples were cooled to 5 °C and stored over 48 h. Motility, viability, membrane functionality, lipid peroxidation and mitochondria activity of the sperm were assessed at 0, 24 and 48 h of storage. Moreover, fertility potential of chilled stored sperm was considered at 24 h of storage. While sperm quality was not affected by LC at the beginning of storage (0 h), supplementation of extender with 1 and 2 mM of LC significantly improved the percentage of sperm motility, viability, membrane integrity and mitochondria activity at 24 h and 48 h compared to other groups. Lipid peroxidation was significantly reduced in sperm samples diluted with 1 and 2 mM LC at 24 h (2.15 ± 0.52 nmol/ml and 2.21 ± 0.52 nmol/ml) and 48 h (3.42 ± 0.49 nmol/ml and 3.38 ± 0.49 nmol/ml) compared to other groups. Furthermore, fertility rates during artificial insemination using sperms cooled for 24 h in the presence of 1 and 2 mM LC were significantly higher (78%) than in the control group (64%). These findings suggest that optimum doses of LC could protect rooster sperm against cool storage-induced functional and structural damages.     

不同组织来源透明质酸的分离纯化及其鉴定

以猪牛眼玻璃体、鸡冠、人脐带为原料,利用水浸提,乙醇沉淀的方法,提取透明质酸粗品,经ＤＥＡＥ－纤维素纯化,获得高纯度的透明质酸．经测定,其蛋白含量０．３８％～０．６３％,分子量７．９×１０~４～４．７×１０~５,收率０．６４％～２．４％,红外扫描图谱与文献报道一致     

Vitrification of the inner perivitelline layer of chicken eggs for use in the sperm-egg interaction assay

The sperm-egg interaction assay is a good predictor of the fertilizing potential of rooster semen; the ability of chicken sperm to interact with the egg can be assessed by counting the number of holes in the inner perivitelline layer (IPVL) of a freshly laid egg. Atlhough isolated IPVL can be stored for up to 24 h, preservation of IPVL for prolonged intervals in liquid nitrogen would facilitate the sperm-egg interaction assay. The objective of this study was to adapt the technique of vitrifying swine oocytes for use with the IPVL. Our hypothesis was that vitrification would not alter the ability of the membrane to bind sperm; therefore, there would be no difference between vitrified and fresh IVPL in the number of hydrolysis holes made by sperm. Our hypothesis was supported; there were no differences in the mean ± SEM number of holes made by the same sample of sperm in vitrified and in fresh membranes (146.0 ± 17.7 holes/mm² IPVL and 159.5 ± 17.7 holes/mm² IPVL, respectively, P > 0.05; n = 123 IVPLs tested). Furthermore, 80% of frozen-thawed membranes were recovered intact. Because vitrification did not significantly change the ability of membranes to bind sperm, vitrified membranes can be safely used for the sperm-egg interaction assay. Vitrified IVPL would ensure availability for sperm evaluation and facilitate wide distribution of IPVL, enabling assays to be conducted even in the absence of facilities or expertise to prepare membranes.     

Supplementation of chilling storage medium with glutathione protects rooster sperm quality

This study was aimed to evaluate the effect of addition of reduced glutathione (GSH) to the extender on the rooster's semen quality parameters and fertility potential. Semen samples were diluted with Lake extender contained 0, 0.5, 1, 2, 4 and 8 mM GSH. Then, were chilled to 5 °C and stored for a period of 48 h. Sperm motion characteristics, viability, membrane integrity, lipid peroxidation, mitochondrial activity and fertility potential were evaluated. At the initiation of the experiment (0 h), GSH did not affect sperm parameters, while 2–4 mM GSH improved (P ≤ 0.05) quality indicators during storage periods. Moreover, the samples treated with 2–4 mM GSH have had a lower lipid peroxidation compared to other groups (P ≤ 0.05). Artificial insemination using the semen samples, which had been stored in groups treated with 2–4 mM GSH for a period of 24 h, led to greater (P ≤ 0.05) fertilizing potential compared to the control group.     

Quercetin supplemented casein-based extender improves the post-thaw quality of rooster semen

The advantageous influence of quercetin (Q) supplementation in an extender has not yet been evaluated for rooster semen cryopreservation. This research was purposely conducted in order to assess the effect of different quercetin concentrations added into an extender on the sperm quality of the rooster subsequent to a freezing-thawing process. After the freezing-thawing process, spermatozoa quality parameters (membrane functionality, acrosome integrity, motility, viability, and abnormal morphology), endogenous enzymes (SOD, CAT, and GPx), mitochondrial activity, DNA fragmentation index, lipid peroxidation (MDA), and ROS were all evaluated. A total of 75 neat pooled ejaculates (3 ejaculates/rooster) were collected from 25 arbor acres roosters (24 wks) twice a week using abdominal massage technique, then divided into five equal aliquots and diluted with an extender containing different doses of Q (CS-Q) as follows: casein extender without Q (control only), casein extender containing 0.040 mg/mL quercetin (CS-Q 0.040), 0.020 mg/mL quercetin (CS-Q 0.020), 0.010 mg/mL quercetin (CS-Q 0.010), and 0.005 mg/mL quercetin (CS-Q 0.005). Our results depicted that adding to the extender with a 0.010 mg/mL Q enhanced (P < 0.01) sperm motility, membrane function, viability, mitochondrial activity, intact acrosome (P < 0.05), SOD (P < 0.001), CAT, and GPx (P < 0.01) compared to the control group at post-thaw. Compared to the control group and other treatment groups after the freeze-thawing process, the addition of 0.005 mg/mL Q into the extender also showed higher (P < 0.05) improvement in the quality of sperm parameters and a higher (P < 0.01) SOD and CAT but did not improve mitochondrial activity and sperm viability. In addition, there was a lower degree of DNA fragmentation index, lower (P < 0.05) lipid peroxidation and ROS in frozen-thawed sperm treated with 0.010 mg/mL and 0.005 mg/mL Q than in control and the other treatment groups. In addition, 0.020 mg/mL Q supplementation into the extender also reduced DNA fragmentation and improved GPx activity compared to the control group at post-thaw. Different concentrations of Q 0.010 and 0.005 mg/mL added to the extender reduced the percentage of abnormal spermatozoa compared to the other groups. The results of this study showed for the first time that the inclusion of an extender with a suitable quercetin concentration of 0.010 mg/mL improved the post-thawed quality of rooster semen.     

Effects of k-carrageenan supplementation or in combination with cholesterol-loaded cyclodextrin following freezing-thawing process of rooster spermatozoa

This experimental research purposely seeks to explore the effect of supplementing k-carrageenan (k-CRG) or CLC (cholesterol-loaded cyclodextrins) or the combined effect of k-CRG and CLC as supplements of antioxidants to an extender for rooster semen freezing. A total of 75 neat pooled ejaculates were collected twice a week from twenty-five (25) commercial line arbor acres broiler roosters (30 wks) during the experimental period. In each replicate, semen samples (n= 15, three ejaculates per rooster) were pooled and divided into nine equal aliquots, and each aliquot was diluted with one of the following extender supplemented with k-CRG, CLC, and k-CRG + CLC after which it was subjected to cryopreservation process using the “pellet” method. In study I, the supplementation of extenders with k-CRG was in five equal aliquots as follows; (0.2, 0.4, 0.6, 0.8) mg/mL and control group (k-CRG 0) mg/mL while in Study II, there was a combination of both k-CRG + CLC (0.4 mg/mL + 1.5 mg/mL, respectively), 0.4 mg/mL k-CRG, 1.5 mg/mL CLC and control group. Sperm quality parameters, endogenous antioxidant enzymes, lipid peroxidation (MDA) and ROS were all assessed after the freeze-thaw process. Our findings in study I indicated that at post-thaw, an optimum 0.4 mg/mL k-CRG supplementation in the extender improved semen quality parameters, endogenous enzymes, MDA and ROS in comparison to the control group. Interestingly prior to the freeze-thaw process, it was depicted in study II that combined k-CRG + CLC (0.4 mg/mL+1.5 mg/mL) inclusion in the extender provided maximum protection to sperm quality parameters, endogenous enzymes, MDA and ROS in comparison to 1.5 mg/mL CLC and control group at post-thaw. Besides, there was also a significant difference observed in the extenders supplemented with combined k-CRG + CLC (0.4 mg/mL +1.5 mg/mL) when compared to 0.4 mg/mL k-CRG for semen quality parameters and endogenous antioxidant enzymes (SOD, CAT, and GPx) but no significant difference was observed for MDA and ROS. Also, there was a significant difference observed in the extender supplemented with 1.5 mg/mL CLC when compared to the control group for semen quality parameters, SOD, CAT, and MDA but no significant difference for GPx and ROS at post-thaw. In conclusion, k-CRG at an optimal dosage of 0.4 mg/mL proved effective for improving post-thaw sperm quality but its combined addition k-CRG + CLC at an optimal concentration of (0.4 + 1.5) mg/mL in the extender provided greater protection to the rooster spermatozoa at post-thaw.     

The behaviour of centrioles and the formation of the flagellum in rooster and drake spermatids

Summary Developmental changes in the formation of the centrioles and flagellum during spermiogenesis in the rooster and drake were studied.Changes in the length and thickness of the wall of the centrioles were observed from an early stage of spermatid development. Before the proximal centriole is attached to the nucleus microtubules were observed near the centrioles joined to them. At this stage of spermatid development changes on the nuclear membrane were observed at a place where the proximal centriole is attached to the nucleus. At the later stage of spermatid differentiation three to five dense extensions in the space of the nuclear invagination and dense bodies or granules near the distal centriole were present. The anterior part of the newly formed flagellum is covered by a cytoplasmic membrane displaying extension which is approximately 1.3 m long. Slight differences between the two species were observed.     

Effect of Achillea millefolium-loaded nanophytosome in the post-thawing sperm quality and oxidative status of rooster semen

The aim of this study was to compare the effectiveness of antioxidants including Achillea millefolium extract (AmE) (n0t1.5: 1.5, n0t3: 3 and n0t4.5: 4.5 mg/L) and AmE loaded in nano phytosome (n1t1.5: 1.5, n1t3: 3 and n1t4.5: 4.5 mg/L) in the freezing of Ross 308 rooster semen. Sperm motility (CASA), membrane integrity (HOS test), viability, total abnormality and enzymatic parameters (SOD, CAT and GPx) were assessed after thawing. AmE-loaded nano phytosome at a concentration of 3 mg/l resulted in significantly (P < 0.05) higher total motility (MOT) (73.78 ± 2.92) and at concentrations of 1.5 mg/L and 3 mg/L in progressive motility (PROG) (14.12 ± 0.38, 16.78 ± 0.38) in comparison with the control group (MOT: 58.48 ± 2.92; PROG: 9.08 ± 0.38). Sperm viability (Vi) was higher (P < 0.05) in n1t3 (74.62 ± 1.55) and membrane integrity (Mi) in n0t3 and n1t3 groups (65.91 ± 1.91, 63.73 ± 1.91, respectively) compared to the control groups (Vi: 66.85 ± 1.55; Mi: 53.18 ± 1.91). Moreover, the lowest percentage of MDA was measured in n1t3 group (1.31 ± 0.31). There was no significant difference for SOD and CAT values with the use of various extenders.In conclusion, we suggest that AmE loaded in nano phytosome at 3 mg/l dose can be added to basic extender for improving rooster sperm motility, viability and oxidative stress values during the freezing procedure.     

Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation

Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 °C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility.