Prepubertal Cats Research Articles

Influence of different warming temperatures on the vitrified testicular fragments from pre-pubertal cat.

Testicular vitrification is an alternative to preserve the genetic material of pre-pubertal animals. However, there are few studies on post-vitrification warming. Hence, the aim was to compare the influence of different warming temperatures on vitrified testicular fragments from pre-pubertal cats. The testicles were fragmented and divided into a control group (non-vitrified) and vitrified, using an association between dimethylsulphoxide and glycerol. The vitrified fragments were warmed at 50, 55 and 60°C/5s. Morphological and morphometric evaluations were carried out using classical histology. Afterwards, the mitochondrial activity was evaluated using Rhodamine 123. The data were expressed in mean and standard error. The differences were considered significant when p<.05. In the histomorphological analysis, the testicular fragment presented seminiferous tubules with poorly developed germinal epithelium, compatible with pre-pubertal animals. The group warmed at 50°C presented similar to the control regarding the maintenance of the integrity of the tubules and cells, without stromal rupture and lamina propria alteration, as well as regarding the maintenance of the junctions between the cells. The group warmed at 55°C showed reduction of the cell junctions, and the one warmed at 60°C had increased detachment of the basement membrane (p<.05). The warming caused a reduction in the tubular diameter inversely proportional and progressive to the increase in temperature, with the highest diameter in the control group and the lowest in the 60°C group. The control group showed a lower incidence of Rhodamine 123, followed in ascending order of the warmings at 55 and 60°C. The higher mitochondrial activity was obtained with 50°C, showing an increase of the metabolic cell function at this temperature. It was concluded that the testicular fragment of pre-pubertal cats presents a better preserved morphology, morphometry and viability when warmed at 50°C.

Initial response of ovarian tissue transcriptome to vitrification or microwave-assisted dehydration in the domestic cat model

BackgroundLong term preservation of living ovarian tissues is a critical approach in human reproductive medicine as well as in the conservation of rare animal genotypes. Compared to single cell preservation, optimization of protocols for tissues is highly complex because of the diversity of cells responding differently to non-physiological conditions. Using the prepubertal domestic cat as a model, the objective was to study immediate effects of vitrification or microwave-assisted dehydration on the global transcriptome dynamics in the ovarian cortex. RNA sequencing was performed on ovarian tissues (n = 6 individuals) from different conditions: fresh tissue after dissection (F), vitrified/warmed tissue (V), tissue dehydrated for 5 min (D5) or 10 min (D10) followed by rehydration. Differential gene expression analysis was performed for comparison pairs V vs. F, D10 vs. F, D5 vs. F and D10 vs. D5, and networks were built based on results of functional enrichment and in silico protein-protein interactions.ResultsThe impact of the vitrification protocol was already measurable within 20 min after warming and involved upregulation of the expression of seven mitochondrial DNA genes related to mitochondrial respiration. The analysis of D10 vs. F revealed, 30 min after rehydration, major downregulation of gene expression with enrichment of in silico interacting genes in Ras, Rap1, PI3K-Akt and MAPK signaling pathways. However, comparison of D5 vs. F showed negligible effects of the shorter dehydration protocol with two genes enriched in Ras signaling. Comparison of D10 vs. D5 showed downregulation of only seven genes. Vitrification and dehydration protocols mainly changed the expression of different genes and functional terms, but some of the differentially expressed genes formed a major in silico protein-protein interaction cluster enriched for mitochondrial respiration and Ras/MAPK signaling pathways.ConclusionsOur results showed, for the first time, different effects of vitrification and microwave-assisted dehydration protocols on the global transcriptome of the ovarian cortex (using the domestic cat as a biomedical model). Acquired data and networks built on the basis of differentially expressed genes (1) can help to better understand stress responses to non-physiological stresses and (2) can be used as directions for future preservation protocol optimizations.

Influence of Microwave-Assisted Drying on Structural Integrity and Viability of Testicular Tissues from Adult and Prepubertal Domestic Cats.

Anhydrous preservation is a promising approach for storage of living biomaterials at nonfreezing temperatures. Using the domestic cat model, the objectives of this study were to characterize changes in histology, DNA integrity, and viability of testicular tissues from adult versus prepubertal individuals during microwave-assisted drying. Testes from each age group were cut into small pieces before reversible membrane permeabilization, exposure to trehalose, and microwave-assisted drying during different time periods. In Experiment 1, water content was monitored for up to 40 minutes of drying. Tissues from adult or prepubertal cats experienced similar decreases of water content during the first 10 minutes. Desiccation progressed slowly between 10 and 20 minutes and then remained stable. In Experiment 2, structural properties were explored at 5, 10, and 20 minutes of desiccation. Percentages of normal seminiferous tubules were lower after 20 minutes drying in adult (43%) than in prepubertal tissues (61%). At the same time point, the proportion of cell degeneration was higher in adult (53%) than prepubertal tissues (28%). Percentages of intact DNA in tissues remained above 85% regardless of the microwave time in both age groups. Lastly, adult and prepubertal tissues only lost 33% of viability in both age groups. Collective results demonstrated for the first time that normal morphology, incidence of degeneration, DNA integrity, and viability of testicular tissues remained at acceptable levels during microwave-assisted drying for 20 minutes. Overall, prepubertal testicular tissues appeared to be more resilient to microwave-assisted desiccations than adult tissues. Importantly, water loss in the presence of trehalose after 20 minutes of desiccation already is compatible with long-term storage of testicular tissues at temperatures above -20°C, which is one step closer to future storage at supra-zero temperatures.

Vitrification of testicular tissue from prepubertal cats in cryotubes using different cryoprotectant associations

Protocols for the cryopreservation of testicular tissue are not yet established. In cats, few studies have been conducted on testicular vitrification using different cryoprotectant associations (CPAs). Thus, the objective of this study was to compare the effect of different CPAs on the vitrification of testicular tissue from prepubertal cats in cryotubes. We used 10 pairs of testicles, with each pair divided into 8 fragments that were distributed into different experimental groups. Two of these fragments were allocated into the control group (CG) and the other six were distributed according to the CPAs to be tested (dimethyl sulfoxide (DMSO)/glycerol (GLY), ethylene glycol (EG)/GLY, or DMSO/EG). The cryoprotectants were used at a final concentration of 5.6 M. The fragments were subjected to vitrification in cryotubes and after 1 week, they were warmed and processed for histomorphologic assessment, quantification of nucleolar organizer regions (NORs), and determination of cell viability. The DMSO/EG and EG/GLY groups presented the greatest cell separation from the cell basement membrane and the highest degrees of retraction of the basal membrane. In these aspects, DMSO/GLY did not differ from the CG and both were significantly superior to the other groups. In terms of cell distinction, visibility of the nucleus, and nuclear condensation, all the vitrified groups had significantly lower values than the CG, while the DMSO/GLY and EG/GLY groups did not differ between themselves. Through the quantification of NORs, the potential for cell proliferation of the CG was found to have a mean of 3.80, while DMSO/GLY presented a mean of 3.60, and thus there was no significant difference between these two groups. The proliferation potentials of both groups were significantly superior to that of the DMSO/EG (mean: 2.07) and EG/GLY (mean: 1.98) groups. In the CG and DMSO/GLY group, 91.8% and 64.2% of cells, respectively, were found to be viable. The cell viabilities of both groups were significantly superior to those of DMSO/EG (52.5%) and EG/GLY (57.10%). Vitrification in cryotubes combined with the use of the DMSO/GLY association was effective in maintaining the histomorphology, cell proliferation potential, and cell viability of testicular tissue from prepubertal cats after cryopreservation.

GnRH-agonist implantation of prepubertal male cats affects their reproductive performance and testicular LH receptor and FSH receptor expression

This study was conducted to investigate the effect of GnRH-agonist implantation in prepubertal tomcats on sexual behavior, reproductive performance, and expression of testicular LH receptor (LHR) and FSH receptor (FSHR) and also to compare the testicular characteristics, LHR and FSHR expression between prepubertal and adult tomcats. In experiment 1, 3-month-old tomcats (n = 6/group) were either treated with or left without 4.7 mg deslorelin implants. Semen collection and evaluation were performed just before castration at 48 weeks after treatment; removed testes were analyzed for mRNA and protein expression of LHR and FSHR. We were able to collect semen from six non-treated cats, whereas in treated cats, semen was uncollectable. The results revealed that sexual behavior was absent in the implanted cats throughout the study period. Testicular volume was found to decrease from 30 weeks after treatment onward in the implanted cats compared to the controls (P < 0.05). Semen production was found only in non-implanted cats. Testicular tissue score, seminiferous tubule diameter, and LHR protein expression were found lower in the implanted cats (P < 0.05), but no differences were observed in mRNA expression of LHR and protein expression of FSHR between groups. The mRNA expression of FSHR was higher in the implanted (P < 0.05) compared to control cats. In experiment 2, testes from prepubertal (n = 6) and adult (n = 6) male cats were collected after castration and analyzed for mRNA and protein expression of LHR and FSHR. No differences were observed in the protein expression of LHR and FSHR between the two groups, whereas mRNA expression of FSHR was higher in prepubertal cats (P < 0.05). Testicular and epididymal weight, diameter of seminiferous tubules, and the testicular grade were higher in the adult compared to prepubertal cats (P < 0.05). In conclusion, deslorelin implants suppressed protein expression of LHR and enhanced mRNA expression of FSHR along with suppression of reproductive function without any adverse effects for at least 48 weeks in male cats.

Long-term contraception in a small implant: A review of Suprelorin (deslorelin) studies in cats.

Rationale:Deslorelin (Suprelorin®; Virbac) is a gonadotropin-releasing hormone (GnRH) agonist licensed in select countries for the long-term suppression of fertility in adult male dogs and male ferrets. This article summarizes studies investigating the use of deslorelin implants for the long-term suppression of fertility in male and female domestic cats.Evidence base:Slow-release deslorelin implants have been shown to generate effective, safe and reversible long-term contraception in male and female cats. In pubertal cats, a 4.7 mg deslorelin implant suppressed steroid sex hormones for an average of approximately 20 months (range 15–25 months) in males and an average of approximately 24 months (range 16–37 months) in females. Reversibility has been demonstrated by fertile matings approximately 2 years post-treatment in both male and female adult cats. In prepubertal female cats of approximately 4 months of age, puberty was postponed to an average of approximately 10 months of age (range 6–15 months) by a 4.7 mg deslorelin implant.Challenges:The large variability in the duration of suppression of gonadal activity makes the definition of the optimal time for reimplantation quite challenging. In addition, the temporary stimulation phase occurring in the weeks following deslorelin implantation can induce in adult female cats a fertile estrus that needs to be managed to avoid unwanted pregnancy. Longer duration and larger scale controlled field studies implementing blinding, a negative control group and a carefully controlled randomization to each group are needed. Furthermore, the effects of repeated treatment need to be investigated. Finally, the effect of treatment on growth and bone quality of prepubertal cats needs to be assessed. However, the ease of use, long-lasting effects and reversibility of deslorelin implants are strong positive points supporting their use for controlling feline reproduction.

184 FOLLICULOGENESIS AND DONOR AGE INFLUENCE MATRIX METALLOPROTEINASE-2 EXPRESSION IN THE DOMESTIC CAT

Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are known to play key roles in the remodelling of extracellular matrix during ovarian folliculogenesis, especially during the final stage of follicle development. To date, little is known about the significance of MMPs and TIMPs during preantral follicle development. This study determined the expression of MMPs and TIMP-1 during various stages of cat folliculogenesis, largely for the purpose of securing information useful to improving in vitro follicle culture. Primordial (~10 follicles/cat), primary (~5 follicles/cat), secondary (~9 follicles/cat), early antral (~9 follicles/cat), and antral (~4 follicles/cat) follicles were physically isolated from ovaries recovered from 15 cats (5 months to 3 years old during follicular stage) undergoing ovariohysterectomy and assessed for expression of MMP-1, -2, -3, -7, -9, and -13 as well as TIMP-1 using real-time quantitative polymerase chain reaction (q-PCR; 2–4 replicates/follicle stage). Additional ovaries were obtained from three prepubertal (6 months old) and three adult (1 year old) cats and ovarian pieces were fixed in Bouin's solution and assessed for MMP-2 and -13 localization using immunohistochemistry. MMP expression data were analysed using the Kruskull-Wallis one-way ANOVA. Follicles from all stages of development expressed MMPs and TIMP-1. Specifically, expression of MMP-2 increased (P &lt; 0.05) as folliculogenesis progressed (10-fold increases from primordial to early-antral and antral stage). There were no differences (P &gt; 0.05) in the expression of other MMPs among follicular classes. For TIMP-1, there was a tendency (P = 0.07) for increased expression after antrum formation (early antral and antral stages). Immunohistochemistry analysis revealed that MMP-2 was expressed in both the oocyte and somatic cells of all follicular stages in prepubertal cats. However, MMP-2 expression was limited to granulosa and theca cells of antral follicles in adult females. MMP-13 was expressed in the granulosa and theca cells of primary, secondary, and antral stage follicles, and there were no differences (P &gt; 0.05) in localization patterns for this protein between prepubertal and adult females. In summary, the study is the first to report the expression of MMPs as well as TIMP-1 in isolated cat follicles. The difference in MMP-2 expression between prepubertal and adult cats suggests that there may be age-specific requirements for in vitro follicle growth. We are keenly interested in this information for underpinning the development of new in vitro microenvironments for growing immature cat follicles. We suspect that such information will be crucial for understanding how to promote the remodelling of the extracellular matrix by creating degradable biomaterials containing MMP-sensitive peptides to allow optimal follicle expansion.

Epidermal Growth Factor (EGF) Sustains In Vitro Primordial Follicle Viability by Enhancing Stromal Cell Proliferation via MAPK and PI3K Pathways in the Prepubertal, but Not Adult, Cat Ovary1

This study examined the influences of epidermal growth factor (EGF) and growth differentiation factor 9 (GDF9) on in vitro viability and activation of primordial follicles in the ovarian tissue of prepubertal (age, <6 mo) versus adult (age, >8 mo) cats. Ovarian cortical slices were cultured in medium containing EGF and/or GDF9 for 14 days. EGF, but not GDF9, improved (P < 0.05) follicle viability in prepubertal donors in a dose-dependent fashion. Neither EGF nor GDF9 enhanced follicle viability in ovarian tissue from adults, and neither factor activated primordial follicles regardless of age group. We then explored how EGF influenced primordial follicles in the prepubertal donors by coincubation with an inhibitor of EGF receptor (AG1478), mitogen-activated protein kinase (MAPK; U0126), or phosphoinositide 3-kinase (PI3K; LY294002). EGF enhanced (P < 0.05) MAPK and AKT phosphorylation, follicle viability, and stromal cell proliferation. These effects were suppressed (P < 0.05) when the tissue was cultured with this growth factor combined with each inhibitor. To identify the underlying influence of age in response to EGF, we assessed cell proliferation and discovered a greater thriving stromal cell population in prepubertal compared to adult tissue. We conclude that EGF plays a significant role in maintaining intraovarian primordial follicle viability (but without promoting activation) in the prepubertal cat. The mechanism of action is via stimulation of MAPK and PI3K signaling pathways that, in turn, promote ovarian cell proliferation. Particularly intriguing is that the ability of cat ovarian cells to multiply in reaction to EGF is age-dependent and highly responsive in prepubertal females.

Effects of different storage protocols on cat testis tissue potential for xenografting and recovery of spermatogenesis

The loss of genetic diversity due to premature death of valuable individuals is a significant problem in animal conservation programs, including endangered felids. Testis tissue xenografting has emerged as a system to obtain spermatozoa from dead immature animals, however protocols to store this tissue before xenografting are still lacking. This study focused on testis tissue cryopreservation and storage from the domestic cat ( Felis catus) classified as “pre-pubertal” and “pubertal” according to spermatogenesis development. Grafts from testis tissue cryopreserved with DMSO 1.4M, recovered after 10 weeks xenografting, presented seminiferous tubules with no germ cells. On the contrary, testis tissue from pre-pubertal animals preserved in ice-cold medium for 2 to 5 days presented no loss of viability or spermatogenic potential, while the number of grafts of pubertal cat testis tissue with germ cells after 10 weeks of xenografting decreased with increasing storage time. Nevertheless, even grafts from pre-pubertal cat testis tissue presented lower anti-DDX4 and anti-BOULE staining (proteins necessary for the meiosis completion), when compared with adult cat testis. Finally, a strong correlation found between testis weight and xenograft outcome may help choose good candidates for xenografting.

The Effect of Ovarian Status and Follicular Diameter on Maturational Ability of Domestic Cat Oocytes

The objective of this study was to clarify the effect of ovarian status and follicular size on morphological normality and maturational ability of cat oocytes. Ovarian status was classified into inactive, follicular, luteal and prepubertal, and follicles were classified into three groups according to their diameter (400-800, 800-1200 and 1200-2000 µm). In each ovarian status, the number of follicles decreased but the percentage of morphologically normal oocytes increased with the growth of follicles (p<0.05). Only a single follicle that was 1200-2000 µm in diameter was observed in two of the five prepubertal cats. In follicles that were 800-1200 µm in diameter, the percentage of normal oocytes and maturation rate were higher in prepubertal cats than in mature cats (p<0.05). Oocyte diameter tended to increase with the growth of follicles. After oocytes were cultured individually in droplets of maturation medium, the oocyte maturation rate increased with the growth of follicles in each ovarian status (p<0.05). In conclusion, oocytes collected from larger follicles possess higher maturational ability in vitro in sexually mature cats. In prepubertal cats, a higher maturation rate can be obtained from oocytes derived from small follicles compared with in mature cats.

Evaluation of Follicular Development and Oocyte Quality in Pre‐pubertal Cats

Our study was conducted to assess the follicular development and availability of sound ovarian oocytes for in vitro production (IVP) of embryos in pre-pubertal cats. The relationship between body and ovarian weight was examined in 93 cats. The results revealed that ovarian weight rapidly increased until 100 days of estimated age. By histological evaluation of ovaries obtained from 11 pre-pubertal cats with estimated age of <20, 20-40 and 100-120 days, it was clarified that the increase in ovarian weight during kitten growth accompanied the increase in the number and size of antral follicles. The follicular diameter and percentage of normal oocytes in secondary/antral follicles also increased as estimated age (body weight) increased. The oocytes obtained from pre-pubertal cats with 100-120 days of estimated age were used for IVP of embryos. The results showed that the success rates of in vitro maturation, in vitro fertilization and development to blastocysts after in vitro culture in pre-pubertal cats were lower than in sexually mature cats. However, the percentage of blastocysts based on the cleaved embryos and cell number of blastocysts in pre-pubertal cats were comparable to those in mature cats. In conclusion, these results suggest that the ovaries of pre-pubertal cats with ≥100 days of age contain oocytes with in vitro developmental competence to blastocysts.