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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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (© 2017 Blackwell Verlag GmbH.)

Cholesterol (cholesterol-loaded cyclodextrins: CLC) treatment of dromedary camel sperm prior to freezing enhances cryosurvival. The present study first validated the efficacy of a heterologous zona-free goat oocyte assay (n=115 oocytes) to evaluate camel sperm function in vitro (Experiment 1: n=6 bulls), then examined the effects of CLC treatment (1.5mg/mL CLC; CLC+) versus no treatment (0 CLC) of fresh (Experiment 2: n=4 bulls) and frozen-thawed (Experiment 3: n=5 bulls) camel sperm to penetrate, de-condense and form pro-nuclei in in vitro-matured goat oocytes. Finally, the ability of fresh 0 CLC and CLC+ sperm to fertilize in vivo was studied by artificially inseminating super-ovulated females (n=7-9 per treatment) and examining embryo production (Experiment 4: n=4-5 bulls/treatment). Camel spermatozoa penetrated (60%) and formed pro-nuclei (33%) in goat oocytes demonstrating the utility of this heterologous system for assessing sperm function in vitro. For fresh spermatozoa, 0 CLC-treated sperm performed better than their CLC+ counterparts for all parameters measured (P<0.05). In contrast, cryopreservation resulted in a sharp decline in sperm-oocyte interaction in 0 CLC aliquots but remained unaltered in CLC+ aliquots demonstrating a protective effect of cholesterol treatment. There was no difference between treatments in the in vitro fertilizing ability of frozen-thawed sperm or in the numbers of embryos retrieved following AI with fresh 0 CLC or CLC+ sperm. We conclude that although CLC treatment of dromedary camel sperm improves sperm motility it fails to confer an advantage to them in terms of improved in vitro sperm-oocyte interaction or in vivo fertilization under the conditions tested., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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).

The cryopreservation of dromedary camel (Camelus dromedarius) sperm has proved challenging with little success reported. The routine application of artificial insemination with frozen semen would assist the flow of valuable genetic material nationally and internationally. The current study sought to examine the effects of cholesterol (cholesterol-loaded cyclodextrin [CLC]) preloading on camel sperm cryosurvival. Ejaculates (n = 3 males; 3 ejaculates per male) were collected using an artificial vagina during the breeding season and extended in HEPES-buffered Tyrode's albumin lactate pyruvate (TALP) and allowed to liquefy in the presence of papain (0.1 mg/mL) before removal of the seminal plasma by centrifugation. Sperm pellets were resuspended (120 million/mL) in fresh TALP and incubated (15 minutes; 37 °C) with 0, 1.5, or 4.5 mg CLC/mL. Sperm suspensions were then centrifuged and reconstituted in INRA-96 containing 20% (v:v) egg yolk and 2.5% (v:v) methylformamide, loaded in 0.5-mL plastic straws, sealed, and cooled for 20 minutes at 4 °C. Straws were frozen over liquid nitrogen (4 cm above liquid; 15 minutes), plunged, and stored. Sperm motility, forward progressive status, and acrosomal integrity were recorded at 0 and 3 hours after thawing and compared with these same parameters before freezing. Aliquots also were stained with chlortetracycline hydrochloride to assess spontaneous sperm capacitation status before freezing and post-thaw. Pretreatment with CLC (1.5 and 4.5 mg/mL) enhanced cryosurvival. Post-thaw sperm motility was highest (P < 0.05) in 1.5 mg CLC/mL immediately after thawing (44%) and after 3 hours incubation at room temperature (34%). Highest post-thaw sperm progressive status was also achieved in the presence of 1.5 CLC. Greater proportions of spermatozoa retained acrosomal membrane integrity when cryopreserved in the presence of CLC, but there was no difference between 1.5 and 4.5 CLC. Although thawed spermatozoa underwent spontaneous capacitation during in vitro incubation, cryopreservation and CLC treatment exerted no effect. In summary, dromedary camel sperm benefit from exposure to CLC before cryopreservation; this may facilitate the routine collection and storage of sperm from this species., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Artificial insemination plays a key role in the genetic management of elephants in zoos. Because freshly extended semen is typically used for artificial insemination in elephants, it has become imperative to optimize conditions for liquid storage and semen transport. The objectives of this study were to examine the interactions between different extenders and storage temperatures on sperm total motility, progressive motility, and acrosomal integrity in Asian (Elephas maximus) and African (Loxodonta africana) elephants. Ejaculates were collected by rectal massage, diluted using a split-sample technique in 5 semen extenders: TL-Hepes (HEP), Modena (MOD), Biladyl (BIL), TEST refrigeration medium (TES), and INRA96 (INR), maintained at 35°C, 22°C, or 4°C. At 0, 4, 6, 12, and 24 hours, aliquots were removed and assessed for sperm total motility, progressive motility, and acrosomal integrity. After 24 hours of storage, African elephant spermatozoa exhibited greater longevity and higher values in sperm quality parameters compared with those of Asian elephants. In both species, semen storage at 35°C resulted in a sharp decline in all sperm quality parameters after 4 hours of storage, whereas storage at 22°C and 4°C facilitated sperm survival. In Asian elephants, MOD and HEP were most detrimental, whereas BIL, TES, and INR maintained motility up to 12 hours when spermatozoa were cooled to 22°Cor4°C. In African elephants, there were no differences among extenders. All media maintained good sperm quality parameters at 22°C or 4°C. However, although MOD, BIL, and INR were most effective at lower temperatures, HEP and TES maintained sperm motility at all storage temperatures. This study demonstrated sperm sensitivity to components of various semen extenders and storage temperatures and offers recommendations for semen extender choices for liquid semen storage for both Asian and African elephants.

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.

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., (Copyright 2005 Wiley-Liss, Inc.)

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.

Measurements of extremely high osmolalities in cauda epididymidal fluids of hibernating bat species led to an investigation of the junctional complex morphology of the epithelium of this sperm storage site. Freeze fracture replicas revealed the presence, at certain times of the year, of a tight junction architecture that resembled that traditionally thought to be exclusive to the blood-testis barrier, the strongest permeability barrier in the body. It is hypothesized that seasonal establishment of these specialized Sertoli cell-like tight junctions is necessary to the maintenance of the high osmotic state of the luminal environment, allowing for the prevention of dilution of its contents by paracellular routes and its protection from bursting under the osmotic pressure contained within.

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.

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.

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.