This study investigated the effects of conditioned medium (CM) from canine amniotic membrane-derived MSCs (cAMSCs) on dog sperm cryopreservation. For this purpose, flow cytometry analysis was performed to characterize cAMSCs. The CM prepared from cAMSCs was subjected to proteomic analysis for the identification of proteins present in the medium. Sperm samples were treated with freezing medium supplemented with 0%, 5%, 10%, and 15% of the CM, and kinetic parameters were evaluated after 4–6 h of chilling at 4 °C to select the best concentration before proceeding to cryopreservation. Quality-related parameters of frozen–thawed sperm were investigated, including motility; kinetic parameters; viability; integrity of the plasma membrane, chromatin, and acrosome; and mitochondrial activity. The results showed that 10% of the CM significantly enhanced motility, viability, mitochondrial activity, and membrane integrity (p < 0.05); however, the analysis of chromatin and acrosome integrity showed no significant differences between the treatment and control groups. Therefore, we concluded that the addition of 10% CM derived from cAMSC in the freezing medium protected dog sperm during the cryopreservation process.

Sperm cryopreservation is a widely used assisted reproductive technology for canine species. The long-term storage of dog sperm is effective for the breeding of dogs living far apart, scheduling the time of artificial insemination that suits the female, and preventing diseases of the reproductive tract. However, spermatozoa functions are impaired during the freeze–thaw processes, which may decrease reproductive performance. Numerous attempts have been made to restore such impairments, including the use of cryoprotectants to prevent the damage caused by ice crystal formation, and supplementation of antioxidants to reduce reactive oxygen species generation due to osmotic stress during the procedure. Egg yolk derivatives, antioxidants, and, more recently, mesenchymal stem cells (MSCs) and their derivatives have been proposed in this research field. This review article will summarize the current literature available on the topic.

Feriel Yasmine Mahiddine, Ahmad Yar Qamar and Min Jung Kim. Journal of Animal Reproduction and Biotechnology 2020;35:268-72. https://doi.org/10.12750/JARB.35.3.268

Although several methods have been developed to improve male fertility and sperm quality, subfertility remains a primary clinical issue in male reproduction worldwide. The aim of this study was to determine the effects of the oral administration of three commensalLactobacillusspp. on healthy normozoospermic dogs and the qualitative parameters of their sperm. Three weeks of supplementation induced a significant decrease of two phyla, Proteobacteria and Tenericutes, and an increase of phylum Firmicutes. At the species level, the number ofFusobacterium perfoetensandAnaerobiospirillum succiniciproducensdecreased, whileLimosilactobacillus reuteriincreased. Parallel to these results, qualitative sperm parameters such as total and progressive motility, acrosome integrity, and other kinematic parameters were significantly enhanced after commensal lactobacilli supplementation. In addition, we showed that Firmicutes were positively correlated with sperm qualitative parameters, while Proteobacteria,F. perfoetens, andA. succiniciproducenswere negatively correlated. Considering the similarities between the gut microbiome of dogs and humans, these results provide more insight into how gut microbiota regulation could improve male sperm quality in both species.

Changes in the gut microbiome can be associated with diseases and affect the overall health of an individual. In the current study, the gut microbiome profile of dogs diagnosed with advanced stages of multicentric lymphoma was compared with that of healthy dogs and analyzed. For this purpose, dogs from veterinary hospitals diagnosed with lymphoma were selected and were further narrowed down to cases of stage IV multicentric lymphoma. Fecal samples from the selected sick and healthy dogs were collected and analyzed using MiSeq sequencing. The gut microbiota in the two groups of dogs was statistically analyzed and compared. The results revealed significant differences in the microbial populations present in sick and healthy dogs. Phylum Actinobacteria and two species (Corynebacterium amycolatum and Streptococcus lutetiensis) were found in high proportions in sick dogs and may be considered as potential biomarkers for canine stage IV multicentric lymphoma. Further investigations need to be conducted to understand the mechanisms they might be involved in.

Canine malignant mammary gland tumors present with a poor prognosis due to metastasis to other organs, such as lung and lymph node metastases. Unlike in human studies where obesity has been shown to increase the risk of breast cancer, this has not been well studied in veterinary science. In our preliminary study, we discovered that leptin downregulated cathepsin A, which is responsible for lysosomal-associated membrane protein 2a (LAMP2a) degradation. LAMP2a is a rate-limiting factor in chaperone-mediated autophagy and is highly active in malignant cancers. Therefore, in this study, alterations in metastatic capacity through cathepsin A by leptin, which are secreted at high levels in the blood of obese patients, were investigated. We used a canine inflammatory mammary gland adenocarcinoma (CHMp) cell line cultured with RPMI-1640 and 10% fetal bovine serum. The samples were then subjected to real-time polymerase chain reaction, Western blot, immunocytochemistry, and lysosome isolation to investigate and visualize the metastasis and chaperone-mediated autophagy-related proteins. Results showed that leptin downregulated cathepsin A expression at both transcript and protein levels, whereas LAMP2a, the rate-limiting factor of chaperone-mediated autophagy, was upregulated by inhibition of LAMP2a degradation. Furthermore, leptin promoted LAMP2a multimerization through the lysosomal mTORC2 (mTOR complex 2)/PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1)/AKT1 (Serine/threonine-protein kinase 1) pathway. These findings suggest that targeting leptin receptors can alleviate mammary gland cancer cell metastasis in dogs.