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Journal: Reproduction, fertility, and development


One important change the head of boar spermatozoa during freeze-thawing is the destabilisation of its nucleoprotein structure due to a disruption of disulfide bonds. With the aim of better understanding these changes in frozen-thawed spermatozoa, two agents, namely reduced glutathione (GSH) and procaine hydrochloride (ProHCl), were added at different concentrations to the freezing media at different concentrations and combinations over the range 1-2mM. Then, 30 and 240min after thawing, cysteine-free residue levels of boar sperm nucleoproteins, DNA fragmentation and other sperm functional parameters were evaluated. Both GSH and ProHCl, at final concentrations of 2mM, induced a significant (P<0.05) increase in the number of non-disrupted sperm head disulfide bonds 30 and 240min after thawing compared with the frozen-thawed control. This effect was accompanied by a significant (P<0.05) decrease in DNA fragmentation 240min after thawing. Concomitantly, 1 and 2mM GSH, but not ProHCl at any of the concentrations tested, partially counteracted the detrimental effects caused by freeze-thawing on sperm peroxide levels, motility patterns and plasma membrane integrity. In conclusion, the results show that both GSH and ProHCl have a stabilising effect on the nucleoprotein structure of frozen-thawed spermatozoa, although only GSH exerts an appreciable effect on sperm viability.

Concepts: Sperm, Spermatozoon, Disulfide bond, Glutathione, Cysteine, Thiol


Egg yolk low-density lipoproteins (LDL) and soybean lecithin were evaluated as replacements for egg yolk in extenders used for the cryopreservation of brown-bear spermatozoa. The motility, viability and acrosomal status of post-thawed spermatozoa were analysed, and an egg-yolk extender was used as a control. The total antioxidant capacity of these extenders was tested. Soybean lecithin showed an effect that was dependent on the soybean concentration (2%, 3.5% and 5%) and source (Type A: 24% -α-phosphatidylcholine, and Type B: 14-23% -α-phosphatidylcholine). Only semen cryopreserved with 5% Type A soybean exhibited a sperm motility similar to that of semen cryopreserved in egg-yolk-based extender after thawing, although the sperm viability and acrosome status were not as high. Semen frozen in an extender containing LDL (10-15%) exhibited improved sperm viability in comparison with the control, but sperm motility was lower. The LDL-based extender exhibited a higher anti-oxidant activity than the egg-yolk extender and soy lecithin-based extenders. The extenders with higher anti-oxidant activity showed improvements in frozen sperm viability but lower semen motility. These results indicate that soybean lecithin did not have the same protective effect as egg yolk during the freezing of brown-bear spermatozoa but suggest that LDL (10-15%) could be a useful substitute for egg yolk in these extenders.

Concepts: Cholesterol, Antioxidant, Sperm, Egg, Spermatozoon, Lutein, Egg yolk, Lecithin


We recently demonstrated that the hollow fibre vitrification (HFV) method (Matsunari et al. 2012) could effectively be applied to the cryopreservation of embryos from diverse species. In this study, we applied the HFV method to the cryopreservation of highly cryosensitive specimens, such as in vitro matured (IVM)/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, as well as IVM/IVF-derived cattle embryos at early cleavage stages. Porcine parthenogenetic morulae (d-4) derived from IVM oocytes were treated with 0.25% pronase to remove zona pellucidae. The resulting blastomeres were isolated from the zona-free morulae by a decompaction treatment followed by gentle pipetting. Bovine IVM-IVF embryos at the 2 to 4 cell (d-1), 8 to 16 cell (d-3), and morula stages (d-5) were then subjected to vitrification. The HFV procedure was performed as described previously using 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5M trehalose as cryoprotectants. Four to twenty embryos, or all of the blastomeres isolated from a single morula, were individually loaded into a cellulose acetate hollow fibre (25mm long, 185μm φ, 15μm membrane thickness) and vitrified. Survival of the vitrified embryos was assessed by in vitro development to blastocysts. Blastomeres recovered after vitrification were aggregated in micro-wells to examine their ability to form blastocysts. The HFV method was demonstrated to be effective for cryopreserving zona-free in vitro-produced porcine morulae and the blastomeres isolated from them (Table 1), as well as bovine IVM-IVF embryos at early cleavage stages. These data demonstrate that the HFV method is effective for highly cryosensitive specimens, such as IVM/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, and IVM/IVF-derived cattle embryos at early cleavage stages. These achievements may expand the technological options in the production of cloned and genetically modified pigs that are useful for biomedical research.

Concepts: Developmental biology, Embryology, Zygote, Ethylene glycol, Cryobiology, Morula, Blastomere, Cryoprotectant


Endemic brucellosis threatens wild herds of wood bison (Bison bison athabascae) in and around Wood Buffalo National Park, the largest genetic reserve of wood bison in the world. The overall goal of our project was to produce and preserve disease-free embryos for the purpose of conserving the genetic diversity of this species. The aim of the present experiment was to determine the effectiveness of washing procedures for removing Brucella bacteria from in vivo-derived wood bison embryos exposed in vitro to the pathogen. Wood bison cows were given 300mg im of Folltropin diluted in 0.5% hyaluronan on the day of follicle wave emergence (Day 0) and 100mg im of hyaluronan on Day 2, and then given 2500IU im of hCG on Day 5 and inseminated 12 and 24h later. Embryos were collected on Day 13. The experiment was done in 6 replicates (n=4 bison/replicate) and an average of 9 embryos/replicate were collected. Zona pellucida-intact embryos were kept in holding medium (PBS+2% fetal calf serum) and transported to a Biosafety Level 3 laboratory at the International Vaccine Centre, University of Saskatchewan. Embryos were transferred through 5 aliquots of holding medium to remove any contaminant before exposure to Brucella. Embryos were divided equally into 2 Petri dishes (representing later wash groups with v. without antibiotics) containing 2.7mL of holding medium (n=2 to 7 embryos per dish/replicate). In a Class II biosafety cabinet, Brucella abortus biovar 1 (1×10(7) to 1×10(9)CFUmL(-1) in 0.3mL) was added to each Petri dish and incubated for 2h at 37°C in 8% CO2. A sample of holding medium was taken before exposure and after incubation for culture as negative and positive controls, respectively. After incubation, embryos in each Petri dish were subjected to a 10-step washing procedure (according to the IETS Manual, 2010) using wash medium (PBS+0.4% BSA) without antibiotics or with antibiotics (100IUmL(-1) of penicillin+100μgmL(-1) of streptomycin). The embryo wash medium was cultured at wash steps 1, 3, 6, and 9. After the tenth wash, the zona pellucida of each embryo was ruptured mechanically using a glass pipette and embryos were cultured individually. Culturing of samples was done on sheep blood agar and specific identification of Brucella organisms was done by PCR. Brucella abortus was detected in 3 embryos from the group washed in medium without antibiotics (3/27), whereas all embryos washed in medium with antibiotics were culture negative (0/27). Brucella abortus was not detected in wash media after the third wash in either group (with or without antibiotics). In summary, Brucella abortus was removed from 89% of in vitro-exposed wood bison embryos using the washing procedure without antibiotics, and from 100% using the washing procedure with antibiotics. Results validate the embryo washing technique for producing Brucella-free wood bison embryos.

Concepts: Fetus, Brucellosis, Agar, Agar plate, American Bison, Bison, Petri dish, Wood Bison


Embryonic morphological classification has great importance for numerous laboratory techniques (from basic to applied research in assisted reproduction). However, the method used to perform the classification of embryos in varying degrees of quality has always been based on the subjectivity of the evaluator. Although quality standards and descriptions of morphological characteristics that categorize an embryo in each grade are established, currently there is not an accurate method that can generate consistent and reliable results. Thus, our work resulted in the development of software able to perform the classification of morphological quality of bovine blastocysts. Artificial intelligence techniques (such as artificial neural networks) were used in the development. Results indicate an overall accuracy of 79.2% in the classification of bovine blastocysts in 3 degrees of quality. For blastocysts classified as excellent or good (class 1), the hit rate is 82.6%; for blastocysts classified as regular (class 2), the hit rate is 16.7%; and for blastocysts classified as poor (class 3), the hit rate is 91.7%.

Concepts: Artificial intelligence, Machine learning, Java, Neural network, Artificial neural network, Connectionism


Asian small-clawed otters (ASCO) are a popular species to exhibit in zoological institutions globally, and are found in managed populations in the United States, Europe, Asia, and Australia. Captive breeding of these otters is integral to population sustainability, with management programs using pedigree analyses to make specific breeding recommendations to ensure long-term genetic viability. Because of the familial social structure of ASCO and limited space within zoos, physical separation of animals is not always possible for temporary breeding prevention, and short-term contraception may be preferred. In US zoos, Suprelorin (deslorelin; Virbac Australia, Milperra, Australia), a GnRH agonist, has been recommended for contraception of female carnivores, due to its small implant size and lack of side effects often associated with hormone-based contraceptives. However, the duration of reproductive suppression with Suprelorin may be excessive and reversibility (i.e. resumption of cyclicity, ovulation, and pregnancies) in implanted females across a range of species has been variable, unpredictable, and prolonged. Since 2006, 49 female ASCO have been implanted with Suprelorin at least once, and, of these, only two females have shown confirmed reversibility with pregnancies. No ASCO females implanted more than once have thus far exhibited reversibility (personal communication, AZA Wildlife Contraceptive Center, St. Louis, MO, USA). In this case study, fecal hormone monitoring of one female ASCO implanted with Suprelorin three times (Dec 2007, Jan 2009, Mar 2010), showed a lack of ovarian cyclicity and ovulation during the three years since her last implant. In an attempt to induce ovarian follicular growth and ovulation, this female was injected (IM) with exogenous gonadotropins (100IU of equine chorionic gonadotropin followed 80h later with 3000IU of porcine luteinizing hormone). Fecal progesterone monitoring confirmed ovulation followed by a 76 day pseudopregnancy, with temporal characteristics similar to those previously reported in naturally cycling ASCO (Bateman et al. 2009 Zoo Biol. 28, 107-126). Following the induced pseudopregnancy and ~55 days of basal progestin levels, this female was observed breeding with a cohabitating ASCO male. Fecal hormone monitoring revealed subsequent ovulation and the occurrence of another pseudopregnancy of normal duration. These preliminary findings suggest that exogenous gonadotropin treatment may be useful for promoting resumption of normal ovarian cyclicity and ovulatory responses in ASCO following prolonged reproductive suppression with Suprelorin.

Concepts: Pregnancy, Luteinizing hormone, Menstrual cycle, Corpus luteum, Human chorionic gonadotropin, Ovulation, Aonyx, Oriental Small-clawed Otter


Breeding by natural mating is ideal for maintaining animal populations. However, the lack of breeding space resulting from an increased number of strains and the decline in fertility caused by inbreeding inhibits the reproduction of subsequent generations. Reproductive technologies, such as gamete preservation and artificial fertilisation, have been developed to overcome these problems. These approaches efficiently produce offspring of laboratory, domestic and wild animals, and can also be used to treat human infertility. Gamete preservation using sperm contributes to improvements in reproductive systems and enables the use of smaller breeding spaces. Although cryopreservation with liquid nitrogen has been used to preserve spermatozoa, freeze-drying without liquid nitrogen, a novel method, facilitates long-term storage of spermatozoa. This method has recently been applied to maintain animal strains. Micro-insemination techniques, such as intracytoplasmic sperm injection (ICSI), are exceptional for improving assisted reproduction. ICSI can be used to fertilise oocytes, even with immotile and immature spermatozoa that are unsuitable for AI and IVF. Reproductive technologies provide a substantial advantage for biobanking and maintaining the genetic diversity of laboratory, domestic and wild animals. This review covers the latest method of sperm freeze-drying and micro-insemination, and future possibilities for maintaining animal strains and populations.

Concepts: Reproduction, Sperm, Reproductive system, Fertility, Spermatozoon, In vitro fertilisation, Assisted reproductive technology, Sexual reproduction


The aim of the present study was to determine whether the use of oocytes from juvenile female mice would improve the efficiency of intracytoplasmic sperm injection (ICSI). In the present study, 15 adult and 14 juvenile C57BL6/J female mice were superovulated, with 17.8 oocytes per mouse harvested from adults, significantly lower than the 40.2 harvested from juveniles (P<0.01). Sixty and 233 oocytes were harvested from C57BL/6J adult and juvenile mice respectively, activated in 10mM SrCl2+5μgmL-1 cytochalasin B for 5-6h and cultured in potassium simplex optimisation medium (KSOM) for 3.5 days, with no differences in morula and blastocyst rates between groups (91.7% vs 96.6%; P>0.05). Twelve hours after injection of human chorionic gonadotrophin, oocytes were harvested from C57BL/6J juvenile mice into KSOM, randomly divided into groups and activated with the same method mentioned above at 0, 2, 4 or 6h and then cultured in KSOM for 3.5 days. There was no significant difference in morula and blastocyst rates among the different groups (P>0.05). Oocytes from juvenile mice activated in 10mM SrCl2 for 2h were subjected to ICSI and the rates of pronuclear formation and Day 1 cleavage were significantly improved compared with the control group (P<0.01). ICSI combined with activation of oocytes from inbred mouse strains (C57BL/6J, C57BL/6N and 129Svev) successfully produced pups. The fertility of some these mice resulting from ICSI was tested, and the animals proved fertile. In conclusion, superovulated juvenile mice can yield more useable oocytes than adult mice, but additional activation is essential for full development of ICSI oocytes harvested from juvenile inbred mice.

Concepts: Present, Fertility, Spermatozoon, Human chorionic gonadotropin, Mouse, Zygote, Morula, Intracytoplasmic sperm injection


The poor captive breeding success of southern hairy-nosed wombats (SHNWs; Lasiorhinus latifrons) has been attributed to the difficulty in accurately characterising oestrous behaviour and their relationship to circulating reproductive hormones. Over two wombat breeding seasons, the use of infrared cameras for 24-h remote behavioural monitoring and the analysis of urine samples collected from seven captive females, were investigated to determine the relationship between behaviour and changes in urinary progesterone metabolites (P4M). Urinary P4M was divided into two concentrations: (1) ≤ baseline P4M values and (2)>baseline P4M values and evaluated against urine volume (mL) and the duration (s) and frequency of 23 behaviours recorded for 8 days surrounding D0 of the luteal phase (D0: a sustained increase in P4M for three or more consecutive days). When P4M was ≤ baseline, the duration of urination and volume both decreased, whereas the duration and frequency of both pacing and rump bites by the female towards the male increased. These results suggest that there were detectable behavioural changes that can be mapped to the changes in the SHNW oestrous cycle, which may be used as behavioural indicators to identify the reproductive status of females.


Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature’s diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (P<0.05). The use of domestic dog oocytes in the cloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones' inheritance of maternal domestic dog mitochondrial DNA.

Concepts: DNA, Dog, Canidae, Jackal, Gray Wolf, Coyote, Canis, Dingo