Single layer centrifugation of fresh dromedary camel semen improves sperm quality and in vitro fertilization capacity compared with simple sperm washing.

Single layer centrifugation (SLC) through a colloid is a tool for selecting viable mammalian spermatozoa but has not been used previously for fresh dromedary camel sperm. Semen from six camels (2 ejaculates/male) was diluted 1:5 (v:v) or 1:10 (v:v) in a Tris-citrate-fructose buffer for mechanical liquefaction by gentle pipetting. Following liquefaction, semen was processed either by SLC or by centrifugation without a colloid (control). Total and progressive motilities, CASA kinematics, vitality and acrosome integrity (eosin-nigrosin) and plasma membrane integrity (Hypo-osmotic swelling test; HOST), and fertilizing ability in a heterologous assay (zona-free goat oocytes) were evaluated. Both total (p = .003) and progressive motilities (p = .003) were higher in SLC-processed than in control semen samples, irrespective of dilution. Positive HOST values increased when using colloid in 1:5 (p = .001) and 1:10 dilution (p = .010). Colloid-selected sperm had higher penetration rates than controls (p < .001 and p = .02 for 1:5 and 1:10 dilutions, respectively). However, only the SLC sperm at 1:5 dilution showed higher percentages of pronuclear formation (p = .02) than controls. Dilution effect was only significant for total motility before in vitro fertilization, with higher values for the 1:5 dilution (p = .033). The recovery rates of motile sperm between dilutions were similar (26.1% vs 35.4%; p = .226). In conclusion, SLC is a promising tool for selecting functional dromedary camel sperm and warrants more research.

Semen collection, ejaculate characteristics and in vitro manipulation of spermatozoa from six species of captive flying-fox (Pteropus spp.).

Seminal characteristics are described in six Pteropus species including the critically endangered P. rodricensis. Spermic ejaculates (~40µL) were collected using electro-ejaculation on 406 of 413 attempts. All flying-fox species had mean percentages of acrosome- and plasma-membrane (PM)-intact spermatozoa of >66% and >73%, respectively; the predominant sperm abnormalities found across all species were damaged, folded or missing acrosomes, bent midpieces and coiled tails. Seminal pH ranged from a low of 7.5 in P. giganteus to a high of 8.2 in P. alecto with the other species in between. Electro-ejaculates recovered in short succession from P. alecto revealed no differences in sperm quality, allowing spermatozoa to be utilised for multi-treatment experiments that evaluated the effects of transportation, incubation temperature and in vitro physico-chemical environments on acrosome and PM integrity. Pteropus alecto spermatozoa were successfully held at ~27°C and 37°C for up to 6h before a reduction in PM integrity (P=0.003) was observed. Acrosome and PM integrity decreased (P<0.000) when P. alecto spermatozoa were incubated at 37°C for 30min in a Tris-citrate buffer of pH 9.0 but remained stable at pH 5.0 to 8.0. Pteropus alecto mean (± s.e.m.) seminal osmolality was 307.0±2.5mOsmkg(-1); nevertheless, spermatozoa were tolerant of media ranging from 160 to 1190mOsmkg(-1) but exposure to media of ≤160mOsmkg(-1) resulted in increased acrosome damage (P<0.000).

Preservation of the spermatozoa of the dromedary camel (Camelus dromedarius) by chilling and freezing: The effects of cooling time, extender composition and catalase supplementation.

This study sought to determine the characteristics of dromedary camel sperm following 24 h chilling and cryopreservation, testing two different buffers and cryoprotectants and the presence of catalase (500 IU/mL). Ejaculates were liquefied in Tris-Citric acid-Fructose buffer, and centrifuged through a colloid. For Experiment 1 (n = 5) sperm were cooled 24 h in Green Buffer or INRA-96® containing 0 or 3% glycerol or ethylene glycol. Experiment 2 (n = 5) used the same six treatments to evaluate sperm cryopreserved after 24 h cooling. A test of fertility was run (n = 12 recipients) with split ejaculates of fresh semen cooled 24 h in Green Buffer with and without glycerol. Experiment 3 (n = 7) cryopreserved sperm cooled 2 and 24 h in Green Buffer without cryoprotectant and with and without catalase. Sperm parameters measured before and after treatments included motility, viability and acrosome integrity. Experiment 1 showed no reduction in all sperm parameters after 24 h and no differences between buffers or presence or not of either cryoprotectant. Experiment 2 showed Green Buffer to be better than INRA for supporting sperm frozen after 24 h cooling while, for both buffers, there were few differences in sperm parameters if cryoprotectant was present or absent. Pregnancies were confirmed in 4/6 animals (67%) while no recipients receiving sperm chilled with glycerol were pregnant. In Experiment 3, catalase-supplemented sperm had maintained better motility 2 h post thaw; there were no differences between 2 or 24 h cooled sperm parameters for presence or absence of catalase. There was neither advantage nor disadvantage to coooling sperm 24 h prior to cryopreservation. We concluded that dromedary sperm can be chilled (24 h) and then either inseminated or cryopreserved. While glycerol presence in Green Buffer during chilling did not interfere with cryosurvival it may be toxic to the fertility of fresh chilled sperm. Catalase supplementation during cooling helps maintain sperm motility post thaw.

Birth of domestic cat kittens of predetermined sex after transfer of embryos produced by in vitro fertilization of oocytes with flow-sorted sperm.

Our goals were to: (1) determine if domestic cat sperm could be sorted to high purity by flow cytometry after overnight shipment of cooled samples; (2) evaluate the efficiency with which sorted sperm could be used to generate cat embryos in vitro; and (3) determine if live kittens of predetermined sex could be produced after transfer of embryos derived by IVF using sorted sperm. Semen samples (n=5) from one male were extended in electrolyte-free solution and shipped overnight at 4 degrees C to the sorting facility. Samples were adjusted to 75x10(6)sperm/mL and stained with Hoechst 33342. After 1h at 34.5 degrees C, samples were adjusted to 50x10(6)sperm/mL with 4% egg yolk TALP+0.002% food dye and sorted by high-speed flow cytometry. Later resort analysis confirmed purities of 94% and 83% for X- and Y-chromosome bearing sperm, respectively. Sorted sperm were centrifuged, re-suspended in TEST yolk buffer and shipped overnight to the IVF laboratory. After IVF of in vivo matured oocytes with X-chromosome bearing sperm, cleavage frequency was 62% (54/87). After IVF of IVM oocytes with control, X- or Y-chromosome bearing sperm, the incidence of cleavage was 42% (48/115), 33% (40/120), and 35% (52/150), respectively, and blastocyst development was 53% (21/40), 50% (11/22), and 55% (23/42), respectively (P>0.05). On Day 2, 45 embryos produced by IVF of in vivo matured oocytes with X-chromosome bearing sperm were transferred to the oviduct of four Day 1 recipients, three of which subsequently delivered litters of one, four, and seven female kittens, respectively. In conclusion, we confirmed that sperm sorting technology can be applied to domestic cats and established that kittens of predetermined sex can be produced.

Collection, seminal characteristics and chilled storage of spermatozoa from three species of free-range flying fox (Pteropus spp.).

This study reports observations on the collection and characteristics of semen from free-range populations of flying fox in Brisbane, Australia. Semen was successfully recovered by electroejaculation from 107 of 115 wild flying foxes (Pteropus alecto, Pteropus poliocephalus and Pteropus scapulatus). A proportion of ejaculates collected from all three species contained seminal vesicle secretions, the incidence of which appeared related to breeding season. Ejaculate volume was small (5--160 microL), requiring a specialised collection vessel and immediate extension to avoid desiccation. Sperm morphological abnormalities and characteristics are described for the first time. In two species (P. scapulatus and P. alecto), sperm quality varied with breeding season. Dilution in Tris-citrate-fructose buffer and subsequent incubation (37 degrees C) of Pteropus semen for 2-3h appeared to have a negative impact on sperm motility and the percentage of sperm with intact plasma membranes and acrosomes and represents a concern for the potential development and use of assisted breeding technology in these species. Preliminary attempts to develop a short-term chilled preservation protocol for flying fox semen revealed that sperm viability (percentage motility and percentage live sperm with intact acrosomes) was significantly reduced after 102 h chilled storage at 5 degrees C; nevertheless, approximately 40% of the spermatozoa were still motile and contained intact acrosomes. Glycerol was neither protective nor detrimental to sperm survival during chilled storage. Microbial flora of the prepuce, urethra and semen of all species were isolated and their antibiotic susceptibility tested. Tetracycline, penicillin, ciprofloxacin, and ceftazidime were the most effective antibiotics in preventing growth of all identified bacteria; however, their effects on sperm survival were not investigated.

Comparative ultrastructure of ant spermatozoa (Formicidae: Hymenoptera).

Mature spermatozoa from spermathecae of founding queens were obtained from 5 species of ants, representing the major subfamilies Myrmicinae (Acromyrmex versicolor, Crematogaster sp.) and Dolichoderinae (Tapinoma sessile, Conomyrma insana, Conomyrma wheeleri). The ultrastructure of ant spermatozoa has many features in common with that of higher insects and is similar to that of other Hymenoptera. Structural similarities to spermatozoa of other Hymenoptera include an acrosome containing an internal rod that extends into the nucleus, two elongate mitochondrial derivatives, a centriolar adjunct, and an axonemal arrangement of 9 + 9 + 2 that includes well-developed coarse, or accessory, tubules. Spermatozoa obtained from A. versicolor, a species that is known to store and utilize viable sperm from this supply for over 10 years, show greater development of the mitochondrial derivatives than do the other species. The most distinctive feature of ant spermatozoa in comparison to other Hymenoptera is the large size of the centriolar adjunct relative to the other organelles. The centriolar adjunct is located posterior to the nucleus, anterior to the mitochondrial derivatives, and opposite the axoneme.

Economical and ecological importance of indigenous livestock and the application of assisted reroduction to their preservation.

Among the many mammalian species that are threatened as the result of habitat destruction are numerous species of rare or little-known native livestock that possess features that render them ideally adapted to their environment. Because of the vital and valuable role many of these species play both to the ecology and economy of their native countries, attention is being directed towards initiating breeding programs that might insure their continued survival. This review introduces and highlights the importance of some of these indigenous species and outlines efforts currently underway to apply assisted reproductive technologies to their conservation.

Reproductive seasonality and the effect of the GnRH agonist deslorelin as a contraceptive in captive male Black Flying-foxes (Pteropus alecto).

Effective contraception would enhance genetic management of captive Pteropus species, which typically breed well in captivity. Male reproductive seasonality was monitored (15-mo interval) in captive P. alecto (6 controls and 5 treated with 4.7 mg deslorelin). In untreated males, there were seasonal changes in testicular volume, body weight and testosterone secretion; testicular volume and body weight peaked in February and March, respectively, whereas testosterone concentration remained >5 ng/ml before rising (P < 0.001) to 24.9 ± 3.6 ng/ml (mean ± SEM) in April. However, there was no corresponding change in sperm quality, and seminal vesicle gland (SVG) secretions remained present in ejaculates. In treated males, testosterone concentration had an initial 'flare' response (mean ± SEM peak: 19.95 ± 3.27 ng/ml) before declining (P < 0.001) by 32 d to basal levels, where it remained. In these males, there was reduced sperm motility after 1 mo (P < 0.001) and the absence of SVG secretions after 4 mo. However, aspermic ejaculates were first recorded 5 mo post-treatment. At 10 mo after treatment, spermatogenesis was still disrupted, when membrane-intact, but non-motile sperm were present in two individuals. Motile sperm were first recovered from one of these males 13 mo after deslorelin treatment. We concluded that captive P. alecto males: (a) had seasonal reproductive changes in testicular volume, body weight and testosterone secretion; (b) produced motile, membrane-intact sperm and SVG secretions throughout the year; and (c) had a rapid decline in testosterone concentration and consequent suppression of testicular function for at least 5 mo following deslorelin administration.

Flow cytometric sorting of fresh and frozen-thawed spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla).

We adapted flow cytometry technology for high-purity sorting of X chromosome-bearing spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla). Our objectives were to develop methodologies for liquid storage of semen prior to sorting, sorting of liquid-stored and frozen-thawed spermatozoa, and assessment of sorting accuracy. In study 1, the in vitro sperm characteristics of gorilla ejaculates from one male were unchanged (P > 0.05) after 8 hr of liquid storage at 15 degrees C in a non-egg yolk diluent (HEPES-buffered modified Tyrode's medium). In study 2, we examined the efficacy of sorting fresh and frozen-thawed spermatozoa using human spermatozoa as a model for gorilla spermatozoa. Ejaculates from one male were split into fresh and frozen aliquots. X-enriched samples derived from both fresh and frozen-thawed human semen were of high purity, as determined by fluorescence in situ hybridization (FISH; 90.7%+/-2.3%, overall), and contained a high proportion of morphologically normal spermatozoa (86.0%+/-1.0%, overall). In study 3, we processed liquid-stored semen from two gorillas for sorting using a modification of methods for human spermatozoa. The sort rate for enrichment of X-bearing spermatozoa was 7.3+/-2.5 spermatozoa per second. The X-enriched samples were of high purity (single-sperm PCR: 83.7%) and normal morphology (79.0%+/-3.9%). In study 4 we examined frozen-thawed gorilla semen, and the sort rate (8.3+/-2.9 X-bearing sperm/sec), purity (89.7%), and normal morphology (81.4%+/-3.4%) were comparable to those of liquid-stored semen. Depending on the male and the type of sample used (fresh or frozen-thawed), 0.8-2.2% of gorilla spermatozoa in the processed ejaculate were present in the X-enriched sample. These results demonstrate that fresh or frozen-thawed gorilla spermatozoa can be flow cytometrically sorted into samples enriched for X-bearing spermatozoa.

Reproduction of the male eastern pipistrelle, Pipistrellus subflavus, in the north-eastern United States.

The major reproductive events in the male eastern pipistrelle, are similar to those of other hibernating vespertilionids. The eastern pipistrelle stores epididymal spermatozoa throughout hibernation, a time when the testes are involuted but accessory gland activity is maintained. However, this species differs from others in that epididymal and testicular spermatozoa persist longer and the weights of the accessory glands are not strongly differentiated between winter and spring/summer. It is suggested that the reproductive period is extended in this species as a function of a more prolonged period of hibernation, resulting in only a brief period of sexual quiescence in mid-summer. The eastern pipistrelle (Pipistrellus subflavus) resembles the canyon bat (P. hesperus) in that some testicular spermatozoa persist during winter. Many aspects of the reproductive anatomy and chronology of these two species are similar; however, eastern pipistrelles apparently lack a seminal vesicle and possess a distinctly different baculum.

Reproductive biology of the male little mastiff bat, Mormopterus planiceps (Chiroptera:Molossidae), in southeast Australia.

The anatomy, biology, and chronology of reproduction in the male of the long penile form of Mormopterus planiceps was studied in southeast South Australia and Victoria. In the morphology of its primary and accessory reproductive organs, M. planiceps was generally reminiscent of other Molossidae; however, in the specialized (sebaceous) nature of the Cowper's gland ducts, in the presence of para-anal glands, and in the unusual, horizontally bifid glans penis and the greatly elongated os penis, it was distinct from other Molossidae studied to date. Young of the year were not reproductively active. Adults displayed a single annual spermatogenic cycle that commenced in spring (September/October) and culminated in spermiogenesis in autumn (February-May), during which period plasma levels of testosterone overtook androstenedione. Thereafter, spermatogenesis appeared to cease (though scattered sperm were seen in the seminiferous tubules until August), but abundant epididymal sperm reserves persisted until September/(October). The accessory glands were hypertrophied during this period, becoming involuted by October. Although the numbers of animals available for study were small, these observations, together with the appearance of spermatozoa in the ductus deferens in August/September suggested that mating could occur during the interval from autumn to spring. Late winter/spring insemination is normal for molossids from temperate environments. However, protracted spermatogenesis commencing in spring that is not accompanied by the availability of spermatozoa until autumn, and a subsequent apparent extension of fertility (epididymal sperm storage, accessory gland hypertrophy) beyond the testicular gametogenic phase, are aspects of the male reproductive cycle in M. planiceps that have not heretofore been described in another molossid bat.

Reproductive biology of the female little mastiff bat, Mormopterus planiceps (Chiroptera: Molossidae) in southeast Australia.

The reproductive biology of the female little mastiff bat (Mormopterus planiceps) was studied from specimens obtained throughout the year in southeast Australia, within the region occupied only by the long penile form of this species. Mormopterus planiceps appeared to undergo a single pregnancy each year and was monotocous. Conception occurred during late winter/early spring after a protracted proestrus, during which the uterine/vaginal epithelia attained an extraordinary thickness; spermatozoa were present in the uterine corpus, vagina, and intramural oviduct for at least 2 months prior to ovulation, although only those present in the oviducts were entire and thus appeared to be viable. Following ovulation, a massive postovulatory infiltration of phagocytes occurred; and the thickness of the uterine corpus epithelium was dramatically reduced. As in other molossids, the tract was bicornuate and dextrally functional. The length of gestation was difficult to determine because early embryonic stages, up to implantation, appeared to span several months (late July/August/September) as did parturition (December/January). Growth of the young was slow; nevertheless, females attained sexual maturity in their first year. Several unusual features included the presence of a long os clitoridis, and tubuloalveolar sudoriferous and associated lobulated, sebaceous, paravaginal glands, which surrounded and emptied into the lower vagina. A deep fornix anterior and lateral to the cervix probably serves to receive the secondary glans penis. The epithelium of the uterine corpus was stratified and indistinguishable, in its cytology and cyclicity, from that of the vagina; furthermore, it lacked a glandular endometrium. This portion of the female tract likely receives the elongated primary glans. These findings are discussed in relation to other Molossidae and to the reproductive biology of male M. planiceps. Although the number of animals sampled was relatively small, the data suggest that this species does not exhibit the usual temperate molossid pattern of late winter/spring coincidence of spermatogenesis and ovulation. It would seem that pregnancy may begin, at least in some individuals, during the inhospitable winter months (when epididymal and uterine spermatozoa are abundant but spermatogenesis has largely terminated) and that additional conceptions continue into the early spring. The occurrence of sperm storage in both sexes of this species is unique among Molossidae studied to date.

Reproductive biology of the male bent-winged bat, Miniopterus schreibersii (Vespertilionidae) in southeast South Australia.

The seasonal chronology of the events of the reproductive cycle, and changes in the structure and function of the primary and accessory organs of the male bent-winged bat, Miniopterus schreibersii, were studied at latitude 37 degrees S in temperate southeastern Australia. The testicular cycle commenced in late spring (November), and sperm appeared in the seminiferous tubules and epididymides in early fall (March). The cycle of the accessory sex gland complex generally paralleled the testicular cycle, reaching maximum hypertrophy at the time of insemination in late fall (April/May). Thereafter, the primary and secondary sex glands (except the ampullary gland) involuted as the animals entered winter torpor. However, a cauda epididymal store of sperm persisted until late spring, and sperm were often observed, as well, in the ampullary gland duct and alveoli throughout winter. This study has confirmed that male Miniopterus differs from other vespertilionids in that accessory gland activity declines following the fall breeding in keeping with the fact that, unlike in other vespertilionids, insemination, ovulation and conception are concurrent events in the fall in this species. The reduced secretory status of the Leydig cells and exceptionally low levels of circulating androgens throughout the year, in combination with the presence of viable epididymidal sperm for most of gestation, are all interesting features of this reproductive cycle.

Reproductive biology of the female leaf-nosed bat, Macrotus californicus, in Southwestern United States: I. a morphometric analysis of the annual ovarian cycle.

In Macrotus californicus (Phyllostomatidae), normal embryogenesis(March-June) is preceded by a period of delayed development (October- March) characterized by implantation and slow growth of the embryo to the primitive streak stage. The events of the annual reproductive cycle can be correlated with ovarian dynamics. Waves of follicular growth appear to be initiated in January and June. Increased multilaminar follicles resulting from the second wave of recruitment appear from August to October. Vesiculation of these follicles is seen in both ovaries from July to January; however, the single Graafian follicle forms only in the right ovary just prior to ovulation in late October-early November. Left ovarian ovulation can be induced by right ovariectomy. High atresia from July to December may retard embryo genesis by failing to provide an optimal hormone milieu for the conceptus. In addition, luteal cells are small during the initial months of embryonic development.The first wave of follicular growth results in the appearance of an increased percentage of growing follicles in April; resultant enhanced estrogen levels may influence the resumption of normal development, an event which also coincides with luteal cell hypertrophy. It would appear possible, therefore,that delayed development in Macrotus is an expression of luteal cell insufficiency and uterine nutritional incompetence resulting from depressed steroid levels. Termination of delay may be brought about by the action of increased levels of estrogen and/or progesterone on the endometrium, perhaps by influencing the activity of mast cells whose products are known in some species to enhance vascularity which in turn could account for added substances essential to normal fetal growth.

Stability of the sperm plasma membrane of hibernating bats (Myotis velifer) compared with other mammals.

Previous experiments have established that the long-lived spermatozoa of hibernating bats are resistant to the acrosome reaction and fertilization in vitro conventional techniques. We tested the hypothesis that the membranes of these spermatozoa are more resistant to perturbation than those of other mammals. We exposed them to non-specific bilayer destabilizing agents and abrupt changes in incubation temperature and tested their response by observing their status (motility and viability) after a time interval compared with other mammals (golden hamster, rabbit, human). The results did not support the hypothesis. The inherent longevity of bat spermatozoa may thus be a function of some component other than unique resilience of their plasma membrane.

Studies on prolonged spermatozoa survival in Chiroptera: a morphological examination of storage and clearance of intrauterine and cauda epididymal spermatozoa in the bats Myotis lucifugus and M. velifer.

The cauda epididymidis, uterine corpus, and cornua and uterotubal junction of Myotis function to retain and preserve normal spermatozoa throughout hibernation. In none of the sites do spermatozoa show features that might account for their extended viability. Spermatozoa stored in the uterus and epididymis show no special orientation toward the epithelium lining these sites, whereas an intimate relationship is established between some sperm and the epithelial cells of the uterotubal junction which might either account for extended postcoital sperm survival or forecast their removal from further participation. Transmission and scanning electron microscopic observations do not disclose any morphological changes in stored luminal spermatozoa. A low rate of phagocytosis of sperm is evident in the female tract during hibernation. However, spermatozoa are evidently not vulnerable to being removed from the storage sites until spring arousal when ovulation occurs. Both uterotubal epithelial cells and phagocytes appear to be involved in the disposal of spermatozoa in the female, whereas epididymal spermatozoa apparently are primarily voided during urination. A mechanism that delays capacitation must underlie the ability of spermatozoa to survive in the female reproductive tract of the hibernating bat.

Cellular composition and steroidogenic capacity of the ovary of Macrotus californicus (Chiroptera: Phyllostomatidae) during and after delayed embryonic development.

In the leaf-nosed bat, Macrotus californicus, a 4.5-month period of delayed early embryogenesis (October-March) precedes a 3.5-month period of normal embryogenesis (March-June). This prolonged gestation provides a unique opportunity to correlate ovarian changes with the events following implantation. The present study investigated luteal cell development and follicular biology during gestation. Circulating progesterone (P) levels following implantation were unchanged before transition to normal development, and were maximal at the start of active gestation. Luteal cell diameters increased during this period. Serum P levels declined prior to parturition, when cells staining positive for 3 beta-hydroxy-5-steroid dehydrogenase-5,4-isomerase (3 beta-HSD) activity were reduced in number and diameter, and enzyme staining was less intense in tissue slices. Subcellular steroidogenic organelles were present during delayed development, but smooth endoplasmic reticulum (SER) was markedly increased after the resumption of normal development at which time also luteal cells reacted positively to staining for 17 beta-HSD. Before parturition, lipid droplet accumulation and reduced SER suggested a reduction in steroid secretion. Large multilaminar follicles stained positive for 3 beta-HSD activity throughout gestation and for 17 beta-HSD except in late delayed development. Thus, the delay in embryogenesis may be due to an inadequately developed corpus luteum or to the steroidogenic activity of the multilaminar follicles.

The status of the corpus luteum during pregnancy in Miniopterus schreibersii (Chiroptera: Vespertilionidae) with emphasis on its role in developmental delay.

Developmental delay is correlated with torpor in the temperate zone bent-winged bat, Miniopterus schreibersii (latitude 37 degrees S) as a period of pre-implantation delay (delayed implantation) followed by a short post-implantation delay (delayed development). During this time, the number of steroidogenic organelles in luteal cytoplasm is greatly reduced compared with normal embryogenesis, and granular endoplasmic reticulum is prominent. Nidation, which occurs while the animals are hibernating, is not accompanied by marked changes in luteal ultrastructure, although the number of lipid droplets decreases somewhat. Progesterone rises slightly but not significantly; however, a pre-nidation decrease in high 17 beta-estradiol levels may play a role in implantation. Following implantation, the conceptus remains delayed at the blastocyst stage for several weeks. During this time the bats remain torpid and the only change in luteal cell ultrastructure is an increase in smooth endoplasmic reticulum as differentiation begins toward the trilaminar stage. At the end of developmental delay hypertrophy of the luteal cell begins and mitochondria and lipid droplets increase, markedly. By this time arousal from hibernation has occurred, placentation takes place and normal development is underway. At placentation, smooth endoplasmic reticulum reaches its maximum in luteal cytoplasm; estrogen and progesterone levels peak about 6 weeks later. For the remaining 2 months of gestation, signs of luteolysis appear. These observations suggest that the corpus luteum of developmental delay, though sub-optimally functional, is prolonged in its luteinization by the arrival of winter when the bats enter torpor. The capacity for maximal steroidogenesis is acquired at the end of winter, some weeks after implantation, when arousal occurs and normal development ensues.

Hyperosmolality and sperm storage in hibernating bats: prolongation of sperm life by dehydration.

Osmolalities of epididymal fluids obtained by micropuncture from hibernating species of bats (Myotis lucifugus) rise during sperm storage periods to as high as 1,523 mmol/kgH2O (approximately 5 times that of plasma). In vitro studies establish that hyperosmolality can preserve viability and prevent initiation of progressive motility in bat epididymal spermatozoa as well as induce their quiescence by reducing respiration. Reduction of osmolality (to 500-600 mmol/kgH2O) induces swelling of sperm and allows the initiation of motility and increased metabolic rate; further reduction of osmolality to < 300 mmol/kgH2O compromises permeability barriers and causes loss of motility. We hypothesize that seasonal establishment of hyperosmotic conditions driven by those cells that constitute the limits of the epididymal lumen dehydrates the compliant spermatozoa and thereby minimizes their metabolic needs. A novel form of cell storage dependent on unique adaptations of the epididymal epithelium for solute and water transport is implicated. To date, the operative osmolyte or osmolytes responsible for elevating osmolality in this system remain elusive.