Sperm Storage Research Articles

Semen Cryopreservation in Oncofertility

Oncofertility, an interdisciplinary field, aims at improving the uality of life of cancer patients prone to iatrogenic infertility by offering fertility preservation options for a potentially fertile future. Semen cryopreservation (SC) is a well-established and effective way of preserving fertility in adolescent and young adult cancer-diagnosed males as they face potential fertility loss due to cancer and its treatments. The pathophysiology of certain cancers, such as testicular cancer or Hodgkin’s lymphoma, has been shown to have an adverse effect on semen uality. Ablative therapies like chemotherapy and radiotherapy can result in compromised sperm parameters depending on the dose and the type of the drug or radiation. Hence, it is recommended to encourage cancer-diagnosed males to store sperm prior to gonadotoxic therapy to restore potential fertility in the future. Despite the feasibility of SC, this option remains underutilised due to several limitations.

The effects of oxidative stress and intracellular calcium on mitochondrial permeability transition pore formation in equine spermatozoa

AbstractThe in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit “classical” features of mPTP; specifically, they are resistant to cyclosporin A‐mediated inhibition of mPTP formation, and they do not require exogenous Ca2+ to form the mPTP. However, chelation of endogenous Ca2+ prevented mPTP formation, indicating a role for intracellular Ca2+ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca2+ stores to form the mPTP in response to low Ca2+ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca2+, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca2+ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity.

The ultrastructure of the spermatheca of Mordellistena brevicauda (Coleoptera, Tenebrionoidea) and the associated bacterial cells

The ultrastructural study on the female reproductive system of the beetle M. brevicauda (Mordellidae) confirmed the positive correlation between the length of the sperm and the size of the female seminal receptacle (Spermatheca).The spermatheca of the species is characterized by an apical bulb-like structure where the spermathecal duct forms numerous folds filled with sperm. At this level many bacterial cells are present intermingled with the duct folds. Some are organized in large structures, such as bacteriomes, while other are single bacteriocytes. The latter are often found near the basal lamina of duct epithelium. In addition, some bacteria are visible in the cytoplasm of the duct epithelial cells. Interestingly, bacterial cells have never been observed in the duct lumen. The possible function of the bacterial cells is discussed.

Experimental heatwaves reduce the effectiveness of ejaculates at occupying female reproductive tracts in a model insect.

Globally, heatwaves have become more common with hazardous consequences on biological processes. Research using a model insect (Tribolium castaneum) found that 5-day experimental heatwave conditions damaged several aspects of male reproductive biology, while females remained unaffected. However, females' reproductive fitness may still be impacted, as insects typically store sperm from multiple males in specialized organs for prolonged periods. Consequently, using males which produce sperm with green fluorescent protein (GFP)-tagged sperm nuclei, we visualized in vivo whether thermal stress affects the ejaculate occupancy across female storage sites under two scenarios; (i) increasing time since insemination and (ii) in the presence of defending competitor sperm. We reconfirmed that sperm from heatwave-exposed males sired fewer offspring with previously mated females and provided new scenarios for in vivo distributions of heat-stress-exposed males' sperm. Sperm from heatwave-exposed males occupied a smaller area and were at lower densities across the females' storage sites. Generally, sperm occupancy decreased with time since insemination, and sperm from the first male to mate dominated the long-term storage site. Reassuringly, although heated males' ejaculate was less successful in occupying female tracts, they were not lost from female storage at a faster rate and were no worse than control males in their offensive ability to enter storage sites occupied by competitor sperm. Future work should consider the potential site-specificity of factors influencing sperm storage where amenable.

Dried storage of sperm at supra-zero temperatures: An alternative for flow cytometric analysis under field conditions

In biotechnological processes such as chromosomal manipulation studies, semen has become a reference in the ploidy verification of the evaluated material. However, the use of fresh samples is limited to the use at field conditions because the analysis is performed under laboratory conditions. Thus, this study aimed to develop a simpler procedure for storing dry semen at 28 °C to reduce cold storage costs. For this, semen samples were evaluated according to established quality semen parameters, a protocol for dry, and 3 sterilization treatments of dry semen were applied to the store. The integrity of the DNA was evaluated every two months, using fresh semen, dry semen (untreated), and particles 3C to compare the peaks by flow cytometry. The results indicated that all samples evaluated before and after drying showed no significant difference in the DNA content. UV-treated semen showed a 1C peak in the histogram up to 180 days of storage and a non-significant difference (P > 0.05) from fresh control in the number of DNA particles up to 120 days and untreated only showed a 1C peak up to 120 days. The developed method may become an interesting procedure to serve as a reference peak for practical flow cytometric analysis, not only in the field of fish biology but also in biomedical and agricultural sciences. Furthermore, dried semen can become a tool for the preservation of genetic material and is a promising low-cost storage technique for biobanking.

Comparative electron microscopy study of spermatozoa in snakes (Lepidosauria, Squamata)

The ultrastructure of snake sperm has received substantial attention primarily because snakes exhibit considerable variability in reproductive characteristics between species, with a wide range of mating systems and reproductive behaviors. Variability of sperm morphology among snake species may be associated with the reproductive strategies of each taxon, such as competition or sperm storage. We provide a detailed description of the sperm ultrastructure of nine snake species (Anilius scytale, Tropidophis paucisquamis, Bothrops jararaca, Oxyrhopus guibei, Dipsas mikanii, Micrurus corallinus, Xenopholis scalaris, Acrochordus javanicus, and Cylindrophis ruffus) and compared this with sperm data from the literature for the following taxa: Liotyphlops beui, Amerotyphlops reticulatus, Trilepida koppesi, Anilios waitii, Anilios endoterus, Aspidites melanochephalus, Boa constrictor amarali, Corallus hortulana, Epicrates cenchria, Boa constrictor occidentalis, Eryx jayakari, Micrurus corallinus, Micrurus surinamensis, Micrurus frontalis, Micrurus altirostris, Oxyuranus microlepidotus, Bothrops alternatus, Bothrops diporus, Crotalus durissus, Agkistrodon contortrix, Vipera aspis, Boiga irregularis, Zamenis schrenckii, Zamenis scalaris, Stegonotus cuculatus, Nerodia sipedon, Liodytes pygaea, and Myrrophis chinensis. We found twelve polymorphic characters in the ultrastructure of sperm among the described snakes. Our work supports the importance of ultrastructural analysis of sperm morphology to understand snake reproduction, and provides sperm-derived morphological characters for phylogenetic analysis.

Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis.

The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl- channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential -60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl- concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.

New species of Rockacestus (Cestoda, Phyllobothriidea) from skates of the genus Bathyraja (Rajiformes, Arhynchobatidae) in the Southwestern Atlantic Ocean with comments on the distribution of the genus

Three new species of Rockacestus Caira, Bueno & Jensen, 2021 were recovered from arhynchobatid skates taken between 37°S–55°S in the Magellanic Province, Southwestern Atlantic Ocean. Rockacestus blasisp. nov. was found in Bathyraja macloviana (Norman, 1937), whereas Rockacestus magellanicussp. nov., and Rockacestus ottavianoisp. nov. were found in Bathyraja magellanica (Philippi, 1902). These species differ from their congeners in having a particular combination of anatomical features, including a moderate to highly folded bothridia, presence of a uteroduct, and a seminal receptacle. Cross-sections of mature proglottids were made for the first time in members of the genus. In addition, the microthrix pattern was described in detail, focusing on the distal bothridial surface, including the apical sucker and marginal loculi resulting in a common microthrix configuration with filitriches and small lanceolate, lingulate, and a particular kind of coniform spinitriches. The diagnosis of Rockacestus is revised to include several features exhibited by the new species. The distribution data of the species currently assigned to Rockacestus are compiled and updated. The finding of R. blasisp. nov., R. magellanicussp. nov., and R. ottavianoisp. nov. not only increases the number of members of Rockacestus in the Magellanic Province in the Southwestern Atlantic from one to four but also expands our knowledge of phyllobothriideans and their association with the softnose skates of the genus Bathyraja Ishiyama, 1958 in the Southern Hemisphere.

Comparative Proteomics Elucidates the Potential Mechanism of Sperm Capacitation of Chinese Mitten Crabs (Eriocheir sinensis).

Sperm capacitation is broadly defined as a suite of biochemical and biophysical changes resulting from the acquisition of fertilization ability. To gain insights into the regulation mechanism of crustacean sperm capacitation, 4D label-free quantitative proteomics was first applied to analyze the changes of sperm in Eriocheir sinensis under three sequential physiological conditions: seminal vesicles (X2), hatched with the seminal receptacle content (X3), and incubated with egg water (X5). In total, 1536 proteins were identified, among which 880 proteins were quantified, with 82 and 224 proteins significantly altered after incubation with the seminal receptacle contents and egg water. Most differentially expressed proteins were attributed to biological processes by Gene Ontology annotation analysis. As the fundamental bioenergetic metabolism of sperm, the oxidative phosphorylation, glycolysis, and the pentose phosphate pathway presented significant changes under the treatment of seminal receptacle contents, indicating intensive regulation for sperm in the seminal receptacle. Additionally, the seminal receptacle contents also significantly increased the oxidation level of sperm, whereas the enhancement of abundance in superoxide dismutase, peroxiredoxin 1, and glutathione S-transferase after incubation with egg water significantly improved the resistance against oxidation. These results provided a new perspective for reproduction studies in crustaceans.

Does morphological maturity anticipate physiological maturity? The pattern of sexual maturity in males of the seabob shrimp Xiphopenaeus kroyeri (Heller, 1862)

The seabob shrimp Xiphopenaeus kroyeri (Heller, 1862) is an important fishing resource and is intensely exploited in South America. Therefore, for the correct and sustainable use of this fishing resource, it is necessary to employ multiple tools to determine the sexual maturity of this species, a key condition for the maintenance of this important economic resource. In this work, we evaluated the morphological (development of secondary sexual characteristics) and physiological (maturation of the reproductive system) sexual maturities in males of X. kroyeri, through the macroscopic and microscopic description of the testes, vasa deferentia and petasma from the juvenile stage to the adult stage. The male reproductive system of seabob shrimp is a paired organ with testes connected to a vasa deferentia, which is anatomically differentiated into three regions: proximal (PVD) (subdivided into anterior, or PVDa, and posterior, or PVDp), medial (MVD) and distal (DVD), the latter region being associated with the terminal ampulla. The morphological maturity of males preceded physiological maturity, meaning the petasma must be formed for the production of seminal fluid and the formation of spermatozoa contained in spermatophores to occur. Petasma formation occurred in the size class 12 ┤13 mm CL, while the production of spermatozoa and spermatophores occurred from 14.1 mm CL. These spermatophores are completely formed in the PVDp and are glycoproteic. Spermatophores are stored in the simple tube of the MVD and DVD. In the DVD, there is an accessory gland forming the ampulla, which produces part of the seminal fluid. The deposition of spermatophores occurs internally in the female’s thelycum with the aid of the petasma, which deposits the seminal fluid in the seminal receptacles or spermathecae. At the end of this process, the thelycum is sealed by a secretion from the accessory gland in the male’s ampulla, forming a sperm plug. Based on the results obtained here, we recommend that only physiologically mature males of X. kroyeri (> 14.1 mm CL) should be exploited by artisanal fisheries and that macroscopic inspection of the ampulla to determine physiological maturity in this species should be avoided, as this region of the DVD has the accessory gland as its main part, a structure responsible for forming the sperm plug, which does not contain spermatozoa or participate in the formation of spermatophores.

Violins we see, species we don’t… Species delimitation of the spider genus Loxosceles Heineken & Lowe (Araneae: Sicariidae) from North America using morphological and molecular evidence

In modern systematics, different sources of evidence are commonly used for the discovery, identification, and delimitation of species, especially when morphology fails to delineate between species or in underestimated species complexes or cryptic species. In this study, morphological data and two DNA barcoding markers—cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2)—were used to delimit species in the spider genus Loxosceles from North America. The molecular species delimitation analyses were carried out using three different methods under the corrected p-distance Neighbor-Joining (NJ) criteria: 1) Assemble Species by Automatic Partitioning (ASAP), 2) General Mixed Yule Coalescent model (GMYC), and 3) Bayesian Poisson Tree Processes (bPTP). The analyses incorporated 192 terminals corresponding to 43 putative species of Loxosceles, of which 15 are newly recognized herein, as putative new species, based on morphology and congruence between molecular methods with COI. The average intraspecific genetic distance (p-distance) was <2%, whereas the average interspecific genetic distance was 15.6%. The GMYC and bPTP molecular methods recovered 65-79 and 69 species respectively, overestimating the diversity in comparison with morphology, whereas the ASAP method delimited 60 species. The morphology of primary sexual structures (males palps and female seminal receptacles) was congruent with most of the molecular methods mainly with COI, showing that they are robust characters for identification at the species level. For species delimitation COI was more informative than ITS2. The diversity of Loxosceles species is still underestimated for North America, particularly in Mexico which holds the highest diversity of this genus worldwide.  

The significance of single-cell transcriptome analysis in epididymis research.

As a crucial component of the male reproductive system, the epididymis plays multiple roles, including sperm storage and secretion of nutritive fluids for sperm development and maturation. The acquisition of fertilization capacity by sperm occurs during their transport through the epididymis. Compared with the testis, little has been realized about the importance of the epididymis. However, with the development of molecular biology and single-cell sequencing technology, the importance of the epididymis for male fertility should be reconsidered. Recent studies have revealed that different regions of the epididymis exhibit distinct functions and cell type compositions, which are likely determined by variations in gene expression patterns. In this research, we primarily focused on elucidating the cellular composition and region-specific gene expression patterns within different segments of the epididymis and provided detailed insights into epididymal function in male fertility.

Do milt storage techniques affect the quality of carp spermatozoa during short-term storage?

Short-term storage and management of milt are crucial parts of hatchery practice. There is speculation that sperm stored in a small Eppendorf could not retain their optimal performance compared to those stored in a 50 ml cell culture flask (CCF), thus raising the question of discrepancy between laboratory and hatchery conditions. Therefore, this study aimed to compare sperm quality parameters, such as osmolality and the ionic composition of seminal plasma, spermatozoa motility and its ability to maintain the volume under various osmotic conditions (310, 230, 150, 90, and 20 mOsm/kg) in fresh sperm and after its storage for 24 h in 1.5 ml Eppendorf or 50 ml CCF at 4 °C without any extender. Freshly collected sperm had higher motility percentages at each osmotic condition than 24 h stored sperm in Eppendorf and CCF. There was no significant difference in spermatozoa motility at various osmotic conditions except 90 mOsm/kg osmolality. Besides, VCL, VSL, VAP, and LIN of spermatozoa, osmolality, and ionic composition of seminal plasma did not significantly differ between these two containers. This study suggested no difference between sperm stored in an Eppendorf or CCF regarding sperm functionality.

Sperm storage organs change with female age and mating history in the Golden Silk spider Trichonephila clavipes (Araneae: Araneidae)

AbstractFemale sperm storage organs in arthropods are used as taxonomic characters since it is assumed that they do not change after maturation. However, in the Golden Silk spider, Trichonephila clavipes, the shape and sclerotization of the spermathecae change with a mating event. Moreover, mating in T. clavipes consists of several hundred copulations, but the male transfers all sperm during the first insertion of each pedipalp. Given this puzzling mating behaviour, we hypothesize that multiple copulations serve purposes other than sperm transfer and uptake, such as further altering the morphology of the storage site and/or transferring substances other than sperm. We compared virgin female T. clavipes with females after two copulations, one for each spermatheca, and after ad‐libitum mating. Each treatment consisted of two sub‐treatments in which the state of the sperm storage organs was inspected after a short (1–3 days) or a long (11 days) time interval after the female's final moult. The size of the spermathecae increased twofold in all mating treatments compared to virgin females. The thickness of the spermathecal wall increased significantly with time in all treatments similarly. The spermathecal epithelium, which releases secretion into the lumen of the spermathecae, seems most active in virgin females during the time when mating would normally occur and turns less active after mating and with age. The spermathecae contain secretion produced by the female before mating; and after mating, sperm and another secretion transferred by the male. The numerous sperm‐less matings in the ad‐libitum treatment resulted in male secretion in the copulatory ducts of the female genitalia, likely impeding sperm transfer by subsequent males. Out study shows that female genitalia can change considerably after having reached maturity and suggests that sperm‐less matings evolved in the context of sperm competition.

Histological Authentication of Reproductive Structures of Little Indian Squid Loliolus (Loliolus) hardwickei, Grey, 1849 (Cephalopoda: loliginidae)

The work deals with the oogenesis, spermatogenesis and associated reproductive structures of the little Indian squid Loliolus (Loliolus) hardwickei. Histological descriptions of the ovary, oviduct, testis and needham sac were made, the various changes observed during maturation were described and the functional significance was discussed. The female reproductive system consists of the ovary, glandular oviduct, lace-like oviducal funnel, paired nidamental gland, accessory nidamental glands and a seminal receptacle for sperm storage on the ventral side of the buccal cavity. The average size of the cells of the ovary varied from 7.635±3.466 µm (primary oogonia) to 1191.114±288.188µm (Ripe oocyte). Oocyte resorption or the presence of atretic oocytes was observed in some sections. The male reproductive system consists of the testis, vas deferens, spermatophoric organ, spermduct, the system of spermatophoric glands (SG) and spermatophoric sac (Needham sac) and penis. The spermatogenesis passed through the differentiation of primodial germ cells, primary and secondary spermatogonia, primary and secondary spermatocytes, rounded spermatids, and elongated spermatids to spermatozoa or sperms. The testis is connected to a thin-walled Needham sac by the spermatophoric duct. The testis is made up of numerous seminiferous tubules and is enveloped by tunica albuginea made up of connective tissue. Histological authentication of reproductive structures in L(L) hardwickei serves the specific purpose of providing a detailed understanding of the species' reproductive biology, which has implications for ecological, environmental, and conservation management efforts.

Characterisation of the urinary microbiome of a frog, and the effect of antibiotics on bacterial abundance and sperm viability during refrigerated storage

Reproductive technologies are increasingly being adopted to improve the conservation management of threatened species. The storage and transport of sperm is a vital aspect of the practical implementation of reproductive technologies, however, bacterial contamination during the storage and transport of sperm samples presents a biosecurity risk and can contribute to a reduction in sperm longevity during storage. The present study: 1) characterised the urinary microbiome (bacterial species composition and abundance) using culture-independent 16S rRNA sequencing; 2) quantified the effect of various doses of gentamicin and streptomycin-penicillin on bacterial abundance (colony-forming units; CFUs) and; 3) quantified the effect of antibiotic supplementation on the sperm viability (proportion live/dead) of spermic urine during a 12-day cold-storage period, using the common eastern froglet, Crinia signifera. Overall, urine samples were found to host a diverse array of bacteria, dominated by the phyla Proteobacteria, Firmicutes, and Bacteroidetes. Bacterial abundance was significantly reduced in all antibiotic treatment groups compared to the control group. Antibiotic supplementation had no effect on sperm viability between day 0 and day 6 of storage, however both antibiotic treatments significantly improved sperm viability from days 9 to 12 of storage compared to the control group. Overall, the results of this study provide novel insight into the urinary microbiome, being the first to use a metagenomics approach to characterise the bacterial community present in the urine of an amphibian. Importantly, this study provides evidence that antibiotic supplementation with either gentamicin or streptomycin-penicillin, minimises bacterial proliferation and improves sperm viability during cold storage. These findings will contribute to the development of biosecurity protocols aimed at reducing the risk of disease transmission and cross-infection from unwanted bacteria and infectious agents in amphibian captive breeding programs.

Male reproductive phenotypes of genetically altered laboratory mice (Mus musculus): a review based on pertinent literature from the last three decades.

Laboratory mice (Mus musculus) are preferred animals for biomedical research due to the close relationship with humans in several aspects. Therefore, mice with diverse genetic traits have been generated to mimic human characteristics of interest. Some genetically altered mouse strains, on purpose or by accident, have reproductive phenotypes and/or fertility deviating from wild-type mice. The distinct reproductive phenotypes of genetically altered male mice mentioned in this paper are grouped based on reproductive organs, beginning with the brain (i.e., the hypothalamus and anterior pituitary) that regulates sexual maturity and development, the testis where male gametes and sex steroid hormones are produced, the epididymis, the accessory sex glands, and the penis which involve in sperm maturation, storage, and ejaculation. Also, distinct characteristics of mature sperm from genetically altered mice are described here. This repository will hopefully be a valuable resource for both humans, in terms of future biomedical research, and mice, in the aspect of the establishment of optimal sperm preservation protocols for individual mouse strains.

Evaluation of Kappa-carrageenan supplementation in extender for post-thaw Kajli ram sperm quality.

Cryopreservation is one of the reliable techniques for long-term storage of sperm. The success of this technique depends on the choice of cryoprotectant; therefore, a plethora of literature has reported the effects of different cryoprotective agents so far. Kappa-carrageenan (κ-carrageenan) is a hydrocolloid polysaccharide extracted from red marine seaweed. Its unique property makes it a promising option as a non-colligative cryoprotectant. The current study aims to evaluate the cryoprotective effect of k-carrageenan along with glycerol on ram sperm quality both after equilibration and freezing. Nine Kajli rams were utilized in this experiment for semen collection through an artificial vagina maintained at 42°C. Qualified samples were diluted in tris egg yolk glycerol (TEYG) extender containing different concentrations of k-carrageenan as 0 mg/mL (control), 0.2, 0.5, 0.8 and 1 mg/mL. Post-thaw assessment was done at 37°C after 24 h of storage, which showed a significant improvement (p < .05) in sperm viability, motility, membrane and acrosome integrity in an extender containing k-carrageenan at a concentration of 0.5 mg/mL compared to control. It is concluded from the current study that the combination of glycerol and 0.5 mg/mL concentration of k-carrageenan improved the sperm post-thaw quality.

Sexual Cycle of the Mediterranean Pond Turtle, &lt;i&gt;Mauremys leprosa&lt;/i&gt; (Testudines: Geoemydidae) in West Central Morocco

We studied the reproductive cycle of male and female Mauremys leprosa in central Morocco. The cycle for males starts in the spring with warming temperatures and the beginning of the dry-season with testicular enlargement and a decrease in epididimydes size accompanied by sperm production. The epididymis contains sperm mainly in winter, spring and summer. Females exhibited follicular enlargement from November reaching a peak in February when a maximum follicle size of 38.5 mm was observed. Ovulation appears to begin in April or May and oviductal eggs were present in our samples from April – July. Suspected mating activity occurred from January – March when environmental temperatures were lowest and the wet-season peaked. Sexual activity was coincident with the onset of maximum storage of sperm in the epididymis. The overall reproductive cycle of this species is similar to that of other Temperate Zone turtles with the exception that mating occurs in the winter, not in the spring and fall. Winter mating may be a response to the wet-season Mediterranean climates. Rain connects isolated pools in ephemeral rivers and streams in Morocco that form during the dry season, thus providing greater opportunities for access to mates.

Fertility and Insemination Characteristics of Sperm Storage Tubules in Old Thai-Native Hens.

We aimed to evaluate the effects of sperm concentration (150-250 × 106 spz/dose) and insemination frequency (once, twice, and thrice weekly) on fertility and sperm storage tubule (SST) characteristics. The SSTs were classified into five categories: namely, SSTs having an unscorable (SST1), empty (SST2), low (SST3), medium (SST4), and high (SST5) sperm count after insemination. The results showed that only insemination frequency affected the fertility rate (p < 0.05). The highest fertility was found in the thrice-weekly insemination group; however, this rate was not significantly different from that for the twice-weekly insemination group, except on day 7, while the once-weekly insemination group showed the lowest fertility rate (p < 0.05) from day four onward. On day 1, the SST characteristics showed no differences among the various insemination frequencies. On day 4, the SST2 and SST3 categories increased in the once-weekly insemination group (p < 0.05), while the SST4 and SST5 categories decreased compared to the twice- and thrice-weekly insemination groups (p < 0.05). On day 7, only the thrice-weekly insemination group maintained a level of SST5 category tubules like that measured on day 1 (p > 0.05). In summary, the insemination dose of 150 × 106 sperm was enough for fertilization, and thrice-weekly insemination was the appropriate frequency in old Thai native hens for maintaining a high sperm density in the SSTs throughout the week.