Characterization of Oviduct Lining, with Emphasis on the Sperm Storage Tubule Region (Uterovaginal Junction), Correlated with Fertility in Mature and Old Thai Native Hens

The effect of age on fertility was investigated in Thai native chickens. The objective of this study was to determine the effects of age (mature and old) on the morphological characteristics of the reproductive organs and the histological characteristics of the uterovaginal junction (UVJ) tissues, resident sperm in the UVJ, and fertility duration in Thai native hens. We found no differences in the morphological characteristics of the reproductive organs, except for the number of follicles and the sizes of the fifth large yellow follicle in mature hens, which were greater than those in old hens (p < 0.05). The diameter of the sperm storage tubules (SSTs) epithelium was larger in old hens than in mature hens (p < 0.05), whereas the epithelium height was lower in old hens (p < 0.05). The number of sperm in the SSTs was greater in mature hens compared with old hens (p < 0.05). Mature hens showed a higher fertility rate than old hens. Our results suggest that, in old hens, the function of the SSTs is impaired, and sperm cannot be retained. Such a deterioration of the SSTs may be one of the factors involved in the decline in fertility.

Gross and Histomorphological Study of the Ovary and Oviduct of Turkey Hen with Especial Emphasis on the Sperm-Host Gland

Turkey bird is one of the popular poultry species which is reared primarily for meat production and considered as one of the major sources of animal protein. With such importance of this species, this study was designed to investigate the gross and histomorphology of the ovary and oviduct with especial emphasis on sperm-host glands of the turkey hen involving ten mature female turkeys (Meleagris gallopavo). The present study highlighted the distribution pattern of sperm-host glands (SHGs) in the oviduct of turkey hen that has a potential role in producing a fertile egg in poultry industries. The oviduct of turkey consists of the infundibulum, magnum, isthmus, uterus, and vagina which are sole distributors for making nutrition enriched egg. The tissue samples were collected from the ovary, different segments of the oviduct and especially uterovaginal junction (UVJ) and infundiomagnal junction of the oviduct. The ovaries and the oviducts were dissected and fixed in Bouins solution and processed for a light microscopic study. Histologically, the left ovary of turkey consisted of an outer cortex and inner medulla, with different stages of follicles. In all areas of the oviduct except the infundibulum and vagina, the tunica mucosa epithelium was lined with ciliated pseudo stratified columnar epithelium, and the lamina propria-submucosa contained branched tubular glands. Sperm-storage tubules were observed in the uterovaginal junction and infundibulo-magnum junction. These tubules were mostly branched, slightly coiled and extended into the lamina propria from the bases of the mucosal folds. These glands had proximal and distal parts; the proximal part was lined by pseudostratified columnar epithelium and distal part by non-ciliated simple columnar epithelium. The number of sperm host glands was more at uterovaginal junction than infundibulomagnal junction. The sperm-host glands might play a functional role in the storage and release of spermatozoa from the SHGs in response to oviposition or ovulation. The results would help poultry scientists and farmers in developing effective disease control and growth strategies.

Avian sperm increase in vitro the release of exosomes from SST-enriched organoids

In birds, oviductal cells play a crucial role in the storage of sperm via cell-to-cell communication including extracellular vesicles (EV). We developed a culture of oviductal organoids enriched in sperm storage tubules (SSTorg) to demonstrate the release of EV. SSTorg were cultured for 24h and added to live (LV), frozen (FZ) and lysed (LY) avian sperm, seminal plasma (SP), avian sperm conditioned medium (CM), or bovine sperm (BV). Western blot demonstrated that SSTorg contained EV protein markers, valosin-containing protein (VCP), heat shock proteins (HSP90A, HSPA8), and annexins (ANXA2, A4, A5). Co-culture with LV significantly decreased the intracellular level of all these proteins except HSPA8. Immunohistochemistry confirmed this result for VCP and ANXA4. LY, CM, SP and BV had no effect on the intracellular level of these proteins, whereas FZ induced a decrease in ANXA2, A4 and A5. In culture media, VCP and HSP90 signals were detected in the presence of LV, FZ, BV, LY, CM and SP, but no ANXA4 signal was observed in the presence of FZ and SP. ANXA2 and A5 were only detected in the presence of LV. The most abundant EV were less than 150 nm in diameter. ANXA4 and A5 were more abundant in EV isolated from the SSTorg culture medium. This study provides a useful culture system for studying interactions between SST cells and sperm. We demonstrated the release of EV by SSTorg in vitro, and its regulation by sperm. This may be of crucial importance for sperm during storage in hens.

Effects of semen dosage, oviductal sperm storage and insemination interval on egg fertility, embryo mortality and hatchability in Nera Black breeder chickens

The biological basis of sustaining fertility in poultry is their ability to store sperm cells in the sperm storage tubules (SST) located in the uterovaginal junction. However, artificial insemination in poultry industry is haphazardly administered in Nigeria without regulation on semen dose and frequency of insemination for optimum fertility. The objective of this study was to establish a semen dosage and insemination interval for maximum fertility and embryonic survival in Nera black layer breeder chickens. A total of 80 breeder hens (52 weeks) were allotted to five (5) treatments with four (4) replicate per treatment. Semen was pooled from 10 matured breeder cocks and inseminated to four groups of hens at varied semen dose of 0.02mL (T1), 0.04mL (T2), 0.06mL (T3) and 0.08mL (T4) of undiluted semen while hens in T5 were mated naturally, both for two consecutive days. 0.02, 0.04, 0.06 and 0.08mL of pooled semen contained 20.43×10 , 40.87×106 , 61.30×106 and 81.74×106 motile spermatozoa. Eggs were collected, stored and artificially incubated weekly for 4 weeks. Fertility, embryo mortality and hatchability parameters were determined. Another 78 breeder hens were allocated into 4 treatments of 5 replicates per treatment with unequal number of hens and were inseminated with 0.02mL of raw semen containing 20.43 × 106 motile sperm cells at 3, 6, 9 and 12 days intervals. Fertility, hatchability and embryo mortality were determined. Data were analysed using descriptive statistics and ANOVA at &0.05 Hatch of fertile eggs in T5 at week 2 (65.36±13.28) was significantly higher (p<0.05) . than T1 (33.83±12.65), T2 (13.25±6.88), T3 (39.17±14.17) and T4 (28.21±11.37). At weeks 1 and 2, there was no significant different across the treatments. Fertility at 4 weeks in T1 (11.53±6.66) was significantly (p<0.05) different from treatments T2 (0.00±0.00), T3 (0.00±0.00), T4 (1.66±1.66) and T5 (0.00±0.00). Total and early embryo mortality in week 3 was significantly higher (p<0.05) in T1 (100.00±0.00, 95.00±5.00) than in treatments T2 (43.75±0.00, 43.75±25.77), T3 (66.67±23.57, 66.67±23.57), T4 (95.00±5.00, 85.00±15.00) and T5 (37.50±23.94, 22.92±15.73). Fertility was significantly (p<0.05) higher in 3 days insemination interval (52.65±7.25) compared with 6 days (39.87±4.70), 9 days (22.98±5.71) and 12 days (36.14±6.89). At weeks 1 and 3, the hatch of fertile eggs across the treatments was not significantly (p>0.05) different from one another. This study suggests that 6 inseminating semen dose of 0.02mL containing approximately 20.43×10 motile sperm cells in Nera black layer breeder chickens will give a maximum fertile period of 5 days, while insemination interval of 3 days using 0.02mLof semen gave highest fertility level.

Proteomic analysis of uterine fluid of fertile and subfertile hens before and after insemination

Avian uterine fluid (UF) and uterovaginal sperm storage tubules (SST) are key components in accepting sperm in SST, maintaining sperm function for several weeks, releasing sperm from SST and their ascent through the uterus. To improve the understanding of sperm storage processes requires investigating UF and SST. This study aimed to identify proteins modulated by sperm in the hen’s genital tract and to highlight their role during sperm storage. Two genetic lines of hens exhibiting long (F+) or short (F−) sperm storage ability were used. GeLC MS/MS analysis was used to establish a quantitative inventory of proteins regulated after insemination in both lines. The proteomic data are available via ProteomeXchange with identifier PXD013514. Immunohistochemistry was used to identify high (ANXA4/ANXA5/OCX32) and low (HSPA8/PIGR) fertility markers in the uterovaginal junction. Our results demonstrated that sperm induced a significant and rapid change in the UF proteomic content and also in the SST epithelium. In F+ hens, mobilization of the ANXA4 protein in the apical part of SST cells after insemination was associated with increased levels of some proteoglycans and binding proteins, and also antimicrobial eggshell matrix protein (OCX32) in the UF. We also observed increased levels of lipid transporters involved in egg formation (VTG1-2, APOA1-4-H). In F− hens, insemination induced increased levels of PIGR in both UF and SST, of ANXA5 in SST, of UF enzymes exhibiting metallopeptidase activity and mucins. In conclusion, sperm induced significant changes in the UF proteomic content. This study also provides evidence that the SST immune system plays a major role in regulating sperm storage.