This study aimed to analyze skimmed milk powder (SMP) and fructose in a new cooling curve to freeze boar semen. A total of 49 semen samples from seven boars were cryopreserved using the new curve with addition of glucose and fructose to the refrigerating diluents: Beltsville Thawing Solution (BTS + G; BTS + F) and Skimmed milk powder (SMP + G; SMP + F), totaling four experimental groups for analysis. To finish the curve, aliquots of semen were packaged in 0.5 ml straws and kept in liquid nitrogen. During the cooling curve, SMP mean spermatic vigor and motility were greater than the BTS (p < 0.05). After thawing, a decrease of spermatic force and motility in both extenders was observed, where the BTS presented spermatic vigor (2.1 ± 0.55) and motility (38 ± 21.8), presenting better results (p < 0.05). There was no statistical difference between sugars added to the BTS and SMP in spermatic force and motility (p > 0.05), although the use of fructose allowed an equalization of motility between the SMP and BTS (p > 0.05). Functionality of membrane was better preserved with the addition of fructose, in both extenders. The rate of sperm viability was significantly higher in extender containing glucose and SMP (71.8 ± 12.5). The percentage of intact acrosome was higher on the treatment containing glucose, independent of the extender (BTS + G: 81.8 ± 7.2, SMP + G: 81.4 ± 14.2). To conclude, the results suggest that the BTS is still the best option to cryopreserve and fructose could be used in boar semen cryopreservation in new cooling curve.

Carnivore semen cryopreservation procedures started with semen washing and centrifuging in culture media for seminal plasma removal and microorganisms elimination. The objective of this study was to perform coatis semen cryopreservation comparing the effects between two extenders Ham’s F-10 and M199 for washing and centrifugation before cryopreservation using Dilutris medium. Semen samples (n = 36) were collected by electroejaculation from six adult male coatis (Nasua nasua) between May and October of 2008 at the Universidade Federal de Mato Grosso Zoo. Sperm total motility (%), progressive sperm motility (0-5), plasma membrane integrity spermatozoa rates (%), and acrosome integrity (%) were analyzed. These fresh semen samples were divided in two fractions, diluted in 1 ml of Ham’s F-10 (Ham’s F-10, Nutricel S.A., Brazil) or M199 (M199, Nutricel S.A., Brazil) and centrifuged at 300 g for 10 min. The supernatant was discarded and pellets resuspended in 1 ml of Dilutris (Dilutris, Minitube®, Brazil), stored at 5ºC for 3 hours, transferred to 0.25 ml straws, placed in liquid nitrogen vapor for 20 min, and immersed in liquid nitrogen. The means/SD for fresh semen and cryopreserved semen using Ham’s F-10/Dilutris and M199/Dilutris were, respectively: 84.28 ± 11.57, 45.38 ± 27.26, and 44.61 ± 25.03 for total motility; 3.64 ± 1.44, 2.15 ± 1.14, and 2.07 ± 1.03 for progressive sperm motility; 92.76 ± 3.46, 84.69 ± 15.77, and 89.76 ± 13.97 for live spermatozoa rate; and 94.76 ± 2.89, 92.35 ± 4.73, and 90.58 ± 7.17 for acrosome integrity. No significant difference (P < 0.05) were observed between the values obtained from the Ham’s F-10/Dilutris or M199/Dilutris treatments. Both treatments demonstrated to be suitable for freezing semen from this species.

The aim of this study was to compare the effects of cryopreservation at approximately -196 °C in liquid nitrogen (N) and freezing at approximately -20 °C in a freezer, on the viability and survival of eight different mastitogenic bacteria inoculated in milk. Bacteria were frozen at approximately -20 °C in a freezer and cryopreserved at approximately -196 °C in liquid nitrogen. An effective preservation method was needed for follow-up samples from cows identified in the South African National Milk Recording Scheme (NMRS) with somatic cell counts above 250 000 cells/mL milk. The organisation responsible for sample collection of the NMRS milk samples also provides producers with liquid nitrogen for their semen flasks at the collection sites. This existing mode of storage and transport could therefore be utilised.Ten samples of each organism were thawed and cultured bi-weekly until week 18 for both temperature treatments. An additional sampling was performed at week 30 for samples frozen at approximately -20 °C. Freezing and cryopreservation did not impair subsequent isolation of Streptococcus dysgalactiae, Streptococcus uberis, Enterococcus faecalis, Staphylococcus aureus (STH) (phage type lytic group III) or Sta. aureus (STA) (phage typed, other than lytic group III). Survival was indicated by the isolation of bacteria from samples, and viability by the strength of growth of the bacteria isolated. The survival of Streptococcus agalactiae decreased after week 12 and Escherichia coli after week 16 of freezing, but both organisms survived under cryogenic preservation until week 18. Coagulase-negative staphylococci survived until week 18 for both freezing and cryogenic preservation.Both storage methods could thus contribute to the improvement of a pro-active approach towards udder health management in South African dairy herds.

With the exception of the domestic cat, all felid species (Felidae) are currently threatened with extinction in their natural habitat. To develop effective and optimal wild cat conservation programmes with assisted reproductive technology (ART) it is necessary to combine advances from different disciplines of science, starting from the biology of the species, through research into the population and habitat, assisted reproductive technologies, establishment of gene banks, developing bioinformatic systems, and ending with biodiversity and endangered species management. In the last few years knowledge of felid reproduction has expanded considerably thanks to comparative studies utilising the domestic cat as a research model for endangered wild cats. Basic reproductive techniques utilised in both domestic cat breeding and rescuing wild felid populations that are threatened with extinction include semen collection and cryopreservation, artificial insemination, oocyte collection, in vitro maturation, in vitro fertilisation, somatic cloning, and embryo transfer. The main directions in which assisted reproductive technologies are being developed in wild cat conservation implementations and the contribution of Polish research centres in advancing these methods are presented.

Introduction The addition of low-molecular-weight antioxidants during the freezing process improves post-thaw sperm quality. The high antioxidant potential of cryopreserved semen could have a positive effect on the motility, viability, and energy status of sperm cells and their ability to bind to the zona pellucida of oocytes. The aim of the study was to determine the effects of different concentrations and combinations of vitamins E and C in a semen extender on selected quality parameters of frozen-thawed canine spermatozoa. Material and Methods The experimental material was the semen of four mixed-breed dogs. Sperm viability (motility, plasma membrane integrity, and mitochondrial function) was examined at 0, 60, and 120 min in semen samples supplemented with the extender and in the controls. Results Combined supplementation with vitamins C + E at a concentration of 200 + 200 μM /1 × 10⁹ spermatozoa had the most profound effect on total sperm motility, linear motility, and the percentage of spermatozoa with intact plasma membrane and active mitochondria. Conclusion The synergistic activity of vitamins E and C had a more beneficial influence on the quality of frozen–thawed sperm than these non-enzymatic antioxidants applied separately.

The therapeutic effect of subcutaneous embedding and revascularisation on the repair of canine bone defects caused by open fracture was examined. A total of 12 adult beagle dogs were randomly split into a control group (group C) and a test group (group T). A section of the radius was removed from each dog under general anaesthesia and the deficit supported by an orthopaedic implant. Group T had the section surgically implanted next to the blood vessel–rich saphenous vein and Group C had it cryopreserved at −80°C. After eight weeks, the bone was surgically implanted back into the matching radial deficit. Bone healing was evaluated by gross morphological and X-ray examinations, post-mortem histology, and successive blood measurements of key bone biochemical markers. At 12 weeks, the bone healing boundary was disappearing more quickly in group T dogs than in their group C counterparts. X-ray and histological examinations showed that the cortical repair of group T subjects was complete and the bony plate arrangement was more regular than that in group C. The levels of bone biochemical markers also proved that the healing state of group T was better. The results showed that the degree of healing, osteoclast activity, and bone formation status of group T were better than those of group C, proving that the vascularised bone graft had a significantly shorter healing time than the cryopreserved bone graft.

Objective: The objective of this study was to determine the effect of adding various doses of green tea extract to the semen of Kacang goats during the sexing process on motility, viability, membrane integrity, malondialdehide (MDA), and deoxyribonucleic acid (DNA) fragmentation. Materials and Methods: It started with the containment of the semen of the Kacang goat, followed by macroscopic and microscopic examinations. If the semen was considered viable, a diluter that had been added with various doses of green tea extract would be added to the semen. After that, sexing was carried out using the percoll gradient density medium. Next, the sexed semen was cryopreserved in liquid nitrogen. Then, an examination of motility, viability, membrane integrity, MDA, and DNA fragmentation was conducted. Result: There was a significant difference between the control and treatment (p &le; 0.05). The highest result was obtained in the treatment of adding 0.05 mg of green tea extract/100 ml of Andromed®. Conclusion: The addition of green tea extract can improve the quality of the sexed semen of the Kacang goat after it has been cryopreserved. [J Adv Vet Anim Res 2022; 9(3.000): 412-418]

Objective: To explore the effect of glycerol at different concentrations using different extenders on DNA fragmentation and motility of frozen-thawed Kintamani Bali dog spermatozoa. Materials and Methods: Sample was collected from four mature Kintamani Bali dogs. Each ejacu¬late was prepared for cryopreservation with two different semen extenders; egg yolk Tris extender and coconut water-based extender. For each extender, three different glycerol concentrations were used; 4%, 6%, and 8%. Each of the six aliquots was loaded into 0.5 ml cryotube, placed on a styrofoam box 5 cm over liquid nitrogen for 10 min, and immersed in liquid nitrogen up to 8 min. Then, the frozen cryotubes were transferred into liquid nitrogen container. The cryotubes were thawed in a water bath at 38.5&#xb0;C for 120 sec. After equilibration and thawing, each sample was assessed for motility parameters and for DNA fragmentation. Results: The addition of 6% glycerol to extenders revealed the most effective addition of glycerol on motility and sperm DNA fragmentation after equilibrium and post-thawing. Conclusion: It is concluded that both extenders with the addition of 6% glycerol are safe to be used as an extender in Kintamani Bali dog semen preservation, and DNA fragmentation of Kintamani Bali dog spermatozoa was not influenced by the freezing procedure. [J Adv Vet Anim Res 2019; 6(2.000): 158-162]

Cryopreservation is very important technique in AI centers of stallions, it preserves sperms for long period and spread the superior genetic merits between different animal’s breeds. However, using of cryopreserved sperms lead to decreasing the fertility between animals, due to lethal damage of sperms during preservation process; so, this study aimed to use the two different freezing media with decreasing post thawing sperm damage. A total of 54 ejaculates were collected from nine pure fertile Egyptian Stallions (6 ejaculates per stallion), individually housed at a Veterinary Clinic in Giza Government. Semen sample was collected by Missouri AV on a regular basis (two collections ⁄ week) during the 2021 breeding season in presence of teaser mare. The collected ejaculates were sent to the laboratory immediately for evaluation by CASA (total concentration, progressive motility, static motility, and sperm abnormalities). Ejaculates were filtrated for removal gel fraction; filtrated ejaculates were diluted by EDTA-glucose media for centrifugation and the resulting sperm pellets were split into 2 equal aliquots and then extended in freezing media. INRA96 Milk-based Extender with glycerol and Egg yolk- based Extender with DMF (dimethylformamide) were used in this study as freezing extenders. Diluted semen was packaged into 0.5 ml straw then cooling at 4 ◦C and freezing by vapor of liquid nitrogen and after that preserved by freezing in liquid nitrogen container at -196⁰ C. After keeping the frozen straws in liquid nitrogen for one week, at least 2 straws were taken for thawing and evaluating post thawing-freezing motility. Finally thawed-frozen semen was inseminated inside fertile mares for calculation the conception rate after one month. Post thawing motility were evaluated in extended semen by two different extenders. The obtained results showed a change in the motility by decreasing in INRA-diluted semen compared to DMF-diluted semen. Conception rate was recorded after insemination and showed a high significant in DMF-diluted semen than INRA diluted semen. We concluded that the frozen semen with DMF based diluent did not decrease the motility of sperms after thawing and achieved high conception rate when compared with INRA based glycerol diluent.

Mesenchymal stem cells derived from bone marrow (BM-MSCs) havethe ability to differentiate into multiple cell lineages. Although the cultivation of these cells has led to a number of characterisation studies, some significant morphological and immunohistochemical properties are still lacking. In this study, isolation of BM-MSCs, morphological features, cell viability, immunophenotypic properties and cryopreservation of BM-MSCs wereexamined in detail. The results demonstrate that the cells isolated from BM-MSCs were plastic adherent and had fibroblastic spindle shape after three passages and get confluent monolayer cells 70-80% after 4-7 days post-subculture. Based on the cell viability analysis, the BM-MSCs showed an increase in cell viability starting from passage 1 until passage 10. Immunophenotypic analysis demonstrated that BM-MSCs were positivefor CD44 and CD105 and negative for CD34. Functional analysis of cryopreservation of BM-MSCs from P6 after 6 months expressed good proliferation rate and cell viability.