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Simple Summary: The dynamic change in gene expression during follicular development is essential for oocyte maturation and ovulation. The genes encoding for estrogen receptor (ESR2) and follicle-stimulating hormone receptor (FSHR) play crucial roles in ovarian follicular development. Gene expression is subjected to different regulatory mechanisms, including microRNAs, which have been demonstrated to regulate specific target genes. No information is currently available on the expression of miRNAs in canine ovarian follicles; therefore, the objective of this study was to evaluate the expression of miRNAs cfa-miR-34a and cfa-let-7c predicted against FSHR and ESR2, respectively, related to their target gene expression in follicular cells throughout the estrous cycle in this species. The genes and miRNAs were evaluated using quantitative PCR analyses. Each miRNA and its target genes were expressed in all estrous phases. A tendency for an inverse relationship was observed between the expression of miR-34a and FSHR only in anestrus, while an inverse correlation was found between miR-let-7c and ESR2 during the entire cycle. The differential expression profile of miR-34a and miR-let-7c and their predicted target genes in canine ovarian follicles obtained in the present study suggests a role of these miRNAs in the negative regulation of FSHR and ESR2 genes throughout the ovarian cycle. The genes encoding for estrogen receptor (ESR2) and follicle-stimulating hormone receptor (FSHR) play crucial roles in ovarian follicular development. This study aimed to determine the expression levels of miRNAs predicted against FSHR and ESR2 mRNAs in follicular cells related to their target genes during the estrous cycle in canines. Antral follicles were dissected from 72 ovaries following ovariohysterectomies. MiRNAs regulating FSHR and ESR2 genes were selected from miRNA databases, and mature miRNA and mRNA expression profiling was performed using real-time polymerase chain reaction (PCR). The best miRNA for each target gene was selected considering the quantitative PCR (qPCR) performance and target prediction probability, selecting only miRNAs with a binding p-value of 1.0, and choosing cfa-miR-34a and cfa-let-7c for FSHR and ESR2, respectively. The expression levels comparing the different phases of the estrous cycle were evaluated using ANOVA. Pearson correlations between the expression pattern of each miRNA and their target genes were performed. Each miRNA and its target genes were expressed in the granulosa cells in all estrous phases. FSHR remained low in anestrus and proestrus, increased (p < 0.05) to the highest level in estrus, and decreased (p < 0.05) in diestrus. ESR2 showed the same trend as FSHR, with the highest (p < 0.05) expression in estrus and the lowest (p < 0.05) in anestrus and proestrus. A tendency for an inverse relationship was observed between the expression of miR-34a and FSHR only in the anestrus phase, while an inverse correlation (r = −0.8) was found between miRNA-7c and ESR2 (p < 0.01). The expression profile of miR-34a and miR-let-7c and their predicted target genes of dog ovarian follicles throughout the estrous cycle observed in this study suggest a role in the transcriptional regulation of FSHR and ESR2, which is the first evidence of the involvement of these miRNAs in the canine follicular function. [ABSTRACT FROM AUTHOR]

Progesterone (P4) concentrations in canines are exceptionally high in the periovulatory period. However, the mechanisms by which P4 modulates final oocyte development in dogs remain to be characterized. The aim of this study was to evaluate the effect of P4 on meiotic development related to the gene expression of connexin 37 (Cx37) and connexin 43 (Cx43) in the canine cumlus oocyte complexes (COCs). COCs were isolated from 120 canine ovaries after a routine ovariohysterectomy. In each experiment, groups of COCs retrieved from the antral follicles were subjected to in vitro maturation (IVM) for 72 h without (control) or with P4 (50 μg/mL and 100 μg/mL) or the P4 receptor antagonist, aglepristone (RU534 at 1 μM and 10 μM). Some of the COCs recovered (from each group) after 72 h of IVM were subjected to meiotic evaluation; the remaining COCs, and those not subjected to IVM, were used to analyze the gene expression of Cx37 and Cx43 by qPCR. The results were evaluated using ANOVA. The addition of P4 increased (P < 0.05) the meiotic development compared to that in the control or aglepristone groups. The highest (P < 0.05) percentage of oocytes in the MII stage was observed upon P4 supplementation. In contrast, the highest percentage (P < 0.05) of oocytes arrested in the GV stage and the lowest (P < 0.05) percentages in the MII stage were observed for COCs cultured with aglepristone. Although a significant decrease in the mRNA levels of both connexins was observed after culturing, no effect on Cx37 and Cx43 gene expression was observed when exogenous P4 was added compared to those of the control group. However, COCs cultured with aglepristone exhibited higher (P < 0.05) expression of Cx37 and Cx43 than COCs in the control IVM-group, regardless of the concentration. In conclusion, our results suggest that a high dosage of P4 during IVM enhances the nuclear maturation of canine oocytes without altering the gene expression levels of Cx37 and Cx43. However, the increase in their expression upon treatment with a P4 antagonist indicates an in vivo role for this hormone in the endogenous modulation of both Cx37 and Cx43. [Display omitted] • High doses of progesterone during IVM may enhance meiotic development. • The addition of progesterone during IVM seems to have no direct effect on Cx37 and Cx43. • The inhibitory effect of aglepristone during IVM suggests an in-vivo role of P4 in Cx37,-43 modulation. [ABSTRACT FROM AUTHOR]

Apoptosis is the cellular mechanism of ovarian follicular atresia in mammals; the aim of this study was to examine the apoptosis‐related cyclic changes in follicular cells of different‐sized antral follicles throughout the oestrous cycle in canines. Ovaries were collected from 26 adult female dogs (1–4 years) following routine ovariohysterectomy. Antral follicles were classified as small, medium or large antral or preovulatory. The percentage of apoptotic cells was determined flow cytometrically using the terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate (dUTP) DNA nick end labelling (TUNEL) assay. Apoptosis rate was quantified as the percentage of TUNEL‐positive cells on a logarithmic scale. Percentages of TUNEL‐positive cells obtained in the flow cytometric assay were compared among oestrous phases and follicular sizes using analysis of variance. Apoptotic follicles were observed in all types of canine follicles in different cycle phases and stages of development, possibly corresponding to the physiological process of the oestrous cycle. Both the oestrous phase and follicular size significantly influenced the apoptosis rate (p <.05). Apoptosis rate increased significantly (p <.05) as follicular development progressed. Apoptosis rate was the highest in large follicles during the oestrous phase (9.2%; p <.05) and the lowest in small follicles during the anestrus period (1.8%; p <.05). In conclusion, our results demonstrate significant differences in the apoptosis rate during the oestrous cycle related to follicle development in the canine ovary. Furthermore, flow cytometry using the TUNEL assay was found to be an effective method for detecting apoptosis in canine follicles. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the expression of connexin (Cx) 37 and Cx43 in canine cumulus–oocyte complexes (COCs) during the oestrous cycle. Cx localisation was analysed by immunohistochemistry and immunofluorescence, whereas protein and gene expression was evaluated by western blotting and quantitative polymerase chain reaction respectively; comparisons were made using analysis of variance. Both Cx37 and Cx43 were expressed in all follicular stages; Cx43 was identified in cumulus cells and Cx37 was identified in cumulus cells, zonae pellucida and oocytes. Immunofluorescence analyses showed that Cx37 remained unchanged during the preovulatory stage but decreased after ovulation, whereas Cx43 remained unchanged before and after ovulation. Cx43 transcripts increased (P < 0.05) during anoestrus and dioestrus in medium-sized follicles but remained unaltered during the pro-oestrus and antral stages during oestrus, before and after ovulation. Cx37 mRNA levels decreased in ovulated COCs (P < 0.05). The highest levels of Cx37 protein (P < 0.05) were detected in the preantral stage during anoestrus. In contrast, strong Cx43 signals were detected in oestrus and in medium-sized antral follicles in dioestrus (P < 0.05). Overall, we demonstrated that Cx37 and Cx43 exhibit different expression patterns, suggesting specific roles throughout growth. Maintenance of Cx expression before ovulation indicates the involvement of Cx37 and Cx43 in the prolonged meiotic arrest. In contrast with other mammals, dogs exhibit a delay in oocyte maturation that has not been fully elucidated. We characterised the pattern of intracellular communication between the oocyte and its companion somatic cell compartment, or cumulus cells, via the expression of connexin 43 and 37 gap junctions. This study advances of understanding of the complex dynamics of dog follicular growth through the connexin gap junctions. Maintenance of connexin expression before ovulation indicates the involvement of connexins 43 and 37 in the prolonged meiotic arrest. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the mRNA LHR and LHR protein expression pattern in the canine ovarian follicles at different stage of development throughout the estrous cycle. Dog ovaries were obtained from 1-6y bitches at proestrus/estrus, anestrus and diestrus stages following ovariohysterectomy. Follicular cells were mechanically recovered from follicles distributed into four types (preantral, small antral, medium antral and large antral). Total RNA extraction was performed and the evaluation of gene expression levels was achieved by relative quantification q-PCR analysis. Intrafollicular amounts of LHR were assessed by western blot method. All results were evaluated by ANOVA. The expression levels of mRNA LHR in follicular cells were observed in every stage of development, however this gene expression varied over the estrous cycle. LHR transcripts increased (P < 0.05) from preantral to antral stage. There were not differences in LHR gene expression among follicles at preantral stages; however, at antral stages the lowest (P < 0.05) LHR mRNA expression was found at anestrus and the highest (P < 0.05) at proestrus/estrus. The LHR protein was also detected in dog follicles in all reproductive phases with patterns varying with stage of follicular development over the reproductive cycle. The antibody against human LHR revealed two bands at ∼90 and ∼67 kDa, probably representing the matured protein and its precursor respectively. Both bands LHR appeared already at preantral follicles increasing (P < 0.05) with growth. A high proportion of LHR was presented as immature forms in all follicles stages during different phases of the estrous cycle. In conclusion, the gene and protein of LHR are differentially expressed in dog follicles over the estrous cycle, increasing with growth and the precursor protein is the most predominant LHR form present in canine follicles. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the expression profiles of growth differentiation factor 9 ( GDF-9 ) and bone morphogenetic protein 15 ( BMP-15 ) mRNA in canine oocytes and follicular cells throughout development at the different phases of the estrus cycle. Ovarian structures (follicles and CL) and plasma progesterone concentration were used to confirm the physiological status of each donor. Denuded oocytes and their follicular cells were recovered from follicles (n = 675) distributed into 4 types (preantral, small antral ∼0.2–0.39 mm, medium antral ∼0.4–5.9 mm, and large antral ∼6–8 mm). Total RNA was extracted and reverse transcribed, and the levels of expression for these 2 genes were determined using a quantitative real-time polymerase chain reaction technique; the data were evaluated by ANOVA. Relative expressions levels of GDF-9 and BMP-15 transcripts were detected in the oocyte and follicular cells in all follicular stages evaluated, showing differential changes (P < 0.05) during development over the estrus cycle. The expression patterns of both transcripts were highly correlated between follicles cells and oocytes (r > 0.8; P < 0.05 for GDF-9 and BMP-15 ), although GDF-9 was expressed at higher levels (P < 0.05) in the oocyte compared with the follicle cells. All cell types showed more GDF-9 mRNA abundance at early developing stages, mainly in the anestrus phase, and declining levels in the later stages (P < 0.05), whereas BMP-15 mRNA levels increased (P < 0.05) in follicular cells and oocytes from the preantral to the later stages, and remained constant during the final preovulatory stage. In conclusion, these two genes were detected in follicular cells and oocytes and were differentially expressed during the follicular development across the estrus cycle. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to characterize in canine oocytes and cumulus cells the dynamic expression of growth differentiation factor 9 (GDF-9) in relation to meiotic development and cumulus expansion throughout in vitro maturation (IVM). Cumulus oocytes complexes (COCs) from ovaries of adult bitches were cultured intact for IVM during 0, 48, 72, and 96 hours. At 0 hours or after IVM, COCs were divided into two groups: one group remained with their cumulus cells and in the other group the cumulus cells were extracted. The expression levels of GDF-9 were determined in both groups using indirect immunofluorescence and Western blot analysis. For immunofluorescence assay, in vivo-matured oocytes collected from oviducts were also used as a positive control. The nuclear stage was analyzed in parallel with 4′-6-diamidino-2-phenylindole staining in denuded oocytes from all maturing groups. The intensity of fluorescence, indicative of GDF-9 expression level, decreased with time (P < 0.05). High expression was observed only in germinal vesicle nonmature oocytes; in contrast, second metaphase oocytes showed only low expression. Western blot analysis showed bands of approximately 56 kd and a split band of approximately 20 kd representing the proprotein and possibly two mature protein forms of GDF-9, respectively. The proprotein was detected in all samples, and it was highly expressed before IVM and in a lesser degree, during the first 48 hours, declining thereafter in coincidence with the expansion of the cumulus cell (P < 0.05). There was a negative correlation (r = −0.97; P < 0.05) between the expression level of GDF-9 and mucification. Mature forms were evident only in COCs, before culture and up to 48 hours of IVM. It was concluded that GDF-9 is expressed in canine oocytes and cumulus cells, mainly in the early developmental states, with low levels in mature oocytes in vitro and in vivo, representing the first approach of GDF-9 dynamic in dog oocyte maturation. [Copyright &y& Elsevier]

Abstract: The aim of this study was to evaluate the effect of sperm cryopreservation and the maturation state of the oocyte on the time course of canine gamete interaction during co-culture for periods of 1–10h. Semen samples were obtained by digital stimulation and ejaculates processed as fresh, chilled and frozen samples. Sperm were co-cultured with immature or in vitro mature bitch oocytes for up to 10h. At hourly intervals, oocytes were evaluated for sperm penetration with epifluorescence microscopy. The results were analyzed statistically using generalized linear models. Spermatozoa treatments had a significant effect on the total percentage of oocyte penetration for both types of oocytes; fresh spermatozoa showed the highest average penetration rate, while frozen sperm showed the lowest value (p <0.05). At the 1st hour of co-culture, chilled and frozen dog sperm had a higher penetration percentage (p <0.05) of in vitro matured canine oocytes (43.6% and 45.7%, respectively) than the fresh sperm had (33.8%). Sperm penetration was directly proportional to the time of incubation, when fresh or chilled sperm were used (P <0.05); in contrast, frozen dog sperm did not change penetration rates with either immature or in vitro matured oocytes over time. There was a significant difference in the average of penetration rate between immature (47.3%) and in vitro matured oocytes (56.6%) throughout the 10h of culturing; irrespective of sperm treatment. The optimal incubation time in terms of maximizing penetration rates probably are dependent on how spermatozoa were processed prior to fertilization. [Copyright &y& Elsevier]

Abstract: The IVM of canine oocytes is characterized by low rates of metaphase II. The objective of this study was to evaluate the effects of hCG on meiotic development of canine oocytes for culture periods up to 96h. Oocytes were collected after ovariohysterectomy. Only oocytes >110μm in diameter, with a homogeneous dark cytoplasm and three or more layers of compact cumulus cells were used. For IVM, the COCs were cultured in TCM-199+10% fetal calf serum, without (medium A control) or supplement with 10IU/mL of hCG (medium B), or with a combination of both media (treatment B/A). The COCs were randomly allocated into three groups. The first and second groups were cultured in either medium A or B, respectively for 24, 48, 72, and 96h. Oocytes of the third group (treatment B/A) were incubated in medium with hCG (medium B) the first 48h and then transferred to medium without hCG (medium A) for an additional 24 or 48h. The proportion of COCs with cumulus cell expansion was also evaluated before fixation. Oocytes were stained with propidium iodide prior to nuclear assessment (with epifluorescence microscopy). COCs with cumulus expansion were evident after 48h of culture. The proportion of COCs with cumulus expansion was higher (P &#60;0.05) for media containing hCG (B or B/A) than for meda lacking hCG (A); this difference was maintained for 72 and 96h in culture. In media A, B and B/A, 23.3, 31.7 and 29.5%, respectively, of oocytes were at metaphase II after 72h, with 20.7, 33.1 and 43.4% at this stage after 96h. The advancement of meiosis was directly proportional to the time of incubation; the highest percentage (P &#60;0.05) of oocytes at metaphase II was observed after 96h of culture when 10IU/mL hCG was present for only the first 48h of culture. [Copyright &y& Elsevier]

The Special Issue features one review and three research articles focusing on mechanisms regulating dog oocyte maturation and development. A review paper presented in this Special Issue summarizes the latest findings on germ cell development, the methods available for obtaining germ cells in vitro, and their potential applications in canid species. Germ cell development: Primordial germ cells are specialized cells that give rise to gametes (sperm and eggs). [Extracted from the article]

Simple Summary: Mammalian ovaries contain many oocytes that are enclosed in different follicles, commonly one oocyte in one follicle. Poly-ovular follicles are those with more than one oocyte within a single follicle. This type of follicle has been observed in many mammals, with an especially high incidence in canines, representing an intriguing condition. The quality of oocytes from poly-ovular follicles is heterogeneous, as different oocyte morphologies have been observed. Oocyte meiotic potential has been a key factor in determining the success rate of the in vitro maturation process in canines because of the mixed population of oocytes collected from follicles at different stages of development and at random phases of the estrus cycle. Several oocytes may be derived from poly-ovular follicles without knowledge of their meiotic capacity in vitro. Therefore, to improve in vitro maturation and reproductive biotechnologies in canines, evaluating the maturity potential of oocytes according to follicle type and estrous cycle is necessary. Our study demonstrated that the efficiency of in vitro maturation of oocytes retrieved from poly-ovular or mono-ovular follicles depends on the follicle type according to the phases of the estrous cycle during collection, which is important to consider when improving in vitro maturation protocols in this species. Poly-ovular follicles are defined as those with more than one oocyte present in single follicles. The occurrence frequency of this follicle type is higher in canines than that in other species. This study aimed to evaluate the in vitro meiotic maturation of dog oocytes from this follicle type in comparison to those from mono-ovular follicles of various sizes (small antral, medium antral, and large antral) considering different phases of the estrus cycle (anestrus, proestrus, estrus, and diestrus). Canine oocytes were obtained separately from the poly-ovular and mono-ovular antral follicles from the ovaries of adult females. In each experimental replicate, cumulus-oocyte complexes (COCs) from poly-ovular and mono-ovular follicles were incubated in supplemented TCM-199 at 38.5 °C and 5% CO2 for 72 h. After culturing, the meiotic development of each oocyte was evaluated using epifluorescence microscopy. Meiotic stages were classified into germinal vesicle (GV), germinal vesicle breakdown (GVBD), first metaphase (MI), and second metaphase (MII). Data were evaluated using an analysis of variance. Oocytes from poly-ovular follicles at all phases exhibited a higher (p < 0.05) percentage of oocytes arrested at the GV stage than those from mono-ovular follicles, showing the highest rate of GV in small antral follicles during anestrus. In contrast, there were no differences in MII rates (p < 0.05) in oocytes from mono-ovular and poly-ovular follicles during the estrus and diestrus phases in all sizes evaluated, with the highest MII rate in estrus. These results suggest that oocytes from poly-ovular follicles can resume meiosis at a slower rate than those from mono-ovular follicles; however, the maturation in vitro of such oocytes is possible. Furthermore, the relationship between the maturation capacity of oocytes from both poly-ovular and mono-ovular follicles depends on the ovarian cycle and follicular development. [ABSTRACT FROM AUTHOR]

Simple Summary: Nowadays, the scientific literature lists several parameters and formulas for the prediction of parturition date in dogs, and their accuracy is affected by various factors: pregnancy phase, litter and also maternal size. The selection of the most appropriate parameter set for ultrasound assessment of gestational age and fetal organ development is essential to predict parturition date with high accuracy, but the high number of factors influencing these parameters challenges their clinical interpretation. In this review, the variables of interest are grouped as (i) maternal parameters, (ii) fetal parameters, (iii) ultrasonographic assessment of maternal and fetal heart rate and blood flow, and (iv) markers of fetal maturity. The aim of this review is to analyze the parameters that predict parturition date in the canine species, especially their accuracy, and finally propose to consider the evaluation of fetal maturity as mark of the readiness for parturition combined with the other most studied parameters. An accurate parturition timing is of key importance for breeders and veterinarians in order to give professional assistance to parturition in dogs. However, pregnancy length calculated from the breeding date has a wide variability. Different parameters and formulas have been described and calculated, as well as their accuracy which is affected by various factors: stage of pregnancy, litter and maternal size. Therefore, the selection of the most appropriate parameter panel poses the challenge of weighing their influences and impact on the overall accuracy. The aim of this review is to analyze the parameters useful for parturition timing, especially their accuracy, and to propose the addition of fetal maturity and criteria for its evaluation to detect readiness for parturition. Parameters, as described in literature, are classified as: (i) maternal parameters, (ii) fetal parameters, (iii) ultrasonographic assessment of maternal and fetal heart rate and blood flow, (iv) parameters indicating fetal maturity. A focus on recently described parameters—such as fetal gastrointestinal motility and fetal lung development detected by quantitative ultrasound—is reported. Currently, the most accurate way to predict parturition day is represented by a prepartum progesterone drop, but the identification of a panel of ultrasonographic parameters combining their significance and their accuracy throughout pregnancy is still needed. [ABSTRACT FROM AUTHOR]

Contents: Growth differentiation factor 9 (GDF‐9) and bone morphogenetic protein 15 (BMP‐15) have pivotal roles in oocyte development in many species, therefore the aim was to investigate these factors during in vitro maturation (IVM) of canine oocytes. Canine cumulus oocytes complexes (COCs) were cultured in six groups for 72 hr in a supplemented TCM199‐Hepes medium as (a) Control group; (b) GDF‐9 antibody (Ab); (c) BMP‐15 Ab; (d) recombinant human (rh) GDF‐9; (e) rh BMP‐15 or (f) rh BMP‐15 and GDF‐9. Data were evaluated by ANOVA. The Abs against GDF‐9 or BMP‐15 had a negative impact on meiotic development. Higher (p < 0.05) number of oocytes was arrested at GVBD stage when they were incubated with either GDF‐9 Ab (64.4 ± 2.1%) or BMP‐15 Ab (67.2%± 4.9%) in comparison to those in control group (32.4 ± 7.8%). In contrast, more (p < 0.05) oocytes in control group reached MI (37.4 ± 1.3%) and MII stages (10.2 ± 2.1%) comparing to those groups with GDF‐9 Ab (23.1 ± 4.7% MI; 0.0% MII) or BMP‐15 Ab (16.4 ± 2.4%MI; 5.9% ± 2.1 MII). Higher rates (p < 0.05) of oocytes in control group stayed still arrested at GV (19.9 ± 8.6%) in comparison to those cultured with either rhGDF‐9 (3.7 ± 0.4%) or rhBMP‐15 (10.9 ± 0.7%). However, there were no differences in MII rates between oocytes cultured with GDF‐9 (14.7 ± 3.1) and BMP‐15 (7.8 ± 2.5) separately. But, more oocytes (p < 0.05) reached the MII stage (20.5 ± 3.8%) compared to those exposed to each protein separately and to the control group. These results suggest that these proteins likely contribute to the meiotic development in dogs. [ABSTRACT FROM AUTHOR]

Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) play important functions in follicular and oocyte development in many species. This study evaluated the dynamic expression of GDF-9 and BMP-15 in canine follicles cells using flow cytometry analysis. Follicular cells were removed from three sizes of antral follicles (small, medium and large) from ovaries of bitches throughout the estrus cycle. Cells were incubated with anti-human GDF-9 polyclonal and anti-mouse BMP-15 monoclonal antibodies. A size and complexity discriminatory gate was used for the cytometryc analysis in the initial dot plot and, additionally, a CD45 marker for leukocyte and propidium iodide (PI) were used for erythrocyte and debris discrimination. The evidence corroborated the presence of both proteins in canine follicle cells, but these proteins were not expressed equally during follicular development. The results analyzed by ANOVA showed that GDF-9 expression decreased ( P < 0.05) during follicular growth in anestrus and proestrous/estrous, but increased in diestrus ( P < 0.05). The expression levels of BMP-15 rose ( P < 0.05) from small to medium sizes in anestrous without changing at diestrus. Small antral follicles expressed the highest values of GDF-9 at anestrus while only BMP-15 showed higher value in small antral follicles at proestrous-estrus compared to diestrus and anestrus. Both proteins decreased in proestrous/estrous ( P < 0.05) with increasing follicle size, registering the lowest levels in large follicles. The flow cytometric assay was able to assess GDF-9 and BMP-15 expression in canine follicular cells, showing that these proteins were differentially expressed during follicular development, possibly related to the special features of canine reproduction. [ABSTRACT FROM AUTHOR]

Simple Summary: The article presents the results of the clinical evaluation and umbilical cord blood analysis obtained from newborn pups delivered by the elective Caesarean section. In human medicine both the umbilical cord blood and Apgar score were proved to provide valuable information on neonatal status. In veterinary medicine very few reports concerning the relation between the clinical neonatal assessment (Apgar score) and umbilical cord blood parameters exist. All puppies show mild respiratory acidemia regardless the Apgar scores result. The lactates were not elevated whereas all the puppies with low Apgar sores had higher glucose and mortality rates. Only pups with low initial Apgar scores were at risk of death within first 24 h. Adaptation to the extra-uterine life is crucial and any practical improvement in neonatal diagnostics and care would be beneficial for newborn puppy survival. The article presents the results of the clinical evaluation (Apgar scores, AS) and umbilical cord blood gas analysis (UCBGA) obtained from clamped umbilical cords of newborn pups delivered by the elective Caesarean section. The study was planned as a controlled clinical study, the newborns were allocated into one of the groups, I—critical neonates (severe distress, AS ≤ 3), II—weak neonates (moderate distress, AS 4–6) and III—healthy neonates (no distress, AS ≥ 7). The following parameters were evaluated: pH (pH units), carbon dioxide partial pressure (pCO2; mmHg), oxygen partial pressure (pO2; mmHg), actual bicarbonate (cHCO3−; mmol/L), total carbon dioxide (cTCO2; mmol/L), base excess of extracellular fluid (BE(ecf); mmol/L), base excess of blood (BE(b); mmol/L), oxygen saturation (csO2; %), lactate (Lac; mg/dL), hematocrit (Hct; %PCV), hemoglobin (cHgb; g/dL), glucose (Glu; mg/dL), ions (Na, K, Ca, Cl). The majority of puppies had low AS at birth (AS 4–6 in 38.1% and AS ≤ 3 in 57.1% of the neonates), but most of them (85.7%) improved by the 20th min. reaching AS of 7 and more. Moreover, puppies with lower AS (≤ 3) were at higher risk of death within the first 24h (20.8% did not survive). The positive correlation was found between Apgar score measured at 0 min and pH (r = 0.46, p = 0.01), and between Apgar score (at 0 min) and base excess in whole blood measured [BE(b)] r = 0.36, p = 0.03). Whereas, a negative correlation was detected between Apgar score at 0 and 5th min and glycemia (r = −0.42, p = 0.05, r = −0.34, p = 0.02 respectively. Overall, the puppies with higher glucose levels had lower Apgar scores and were at higher risk of death. Furthermore, in our study, the newborn puppies had mild acidemia with elevated pCO2 levels and the HCO3 at the lower range of normal limits, suggesting the mixed component in the acidemic state. Adaptation to extra-uterine life is crucial and any practical improvement in neonatal diagnostics and care would be beneficial for newborn puppy survival. [ABSTRACT FROM AUTHOR]

Simple Summary: In vitro production of canine embryos is a technique that can be used as a model to conserve endangered species and to establish efficient breeding systems for domestic dogs. However, compared with other species, the success rates of in vitro embryo production (IVEP) in dogs are low. L-Carnitine (LC) is a small water-soluble molecule; it plays an essential role in fatty acid metabolism and acts as a potent antioxidant. Various studies have reported the beneficial impacts of LC on IVEP in many mammalian species other than dogs. Therefore, these experiments investigated the effects of LC supplementation during in vitro maturation (IVM) on canine oocytes maturation, fertilization, and development in vitro. We show that the supplementation of IVM media with LC has positive impacts on oocyte maturation, fertilization, and preimplantation embryo development rates. We also demonstrate that 0.6 mg/mL LC is the most beneficial concentration to be used. It resulted in significantly higher maturation, fertilization, and embryo developmental rates than the control and other LC concentrations. These outcomes are essential for refining the IVM conditions that can advance the efficiency of assisted reproductive technologies (ARTs) in dogs. This study aimed to investigate the effect of L-Carnitine (LC) supplementation during in vitro maturation (IVM) of canine oocytes on nuclear maturation, fertilization status, and preimplantation development. Cumulus–oocyte complexes (COCs) collected from the ovaries of ovariohysterectomized female dogs were matured in vitro for 72 h in a TCM-199 medium supplemented with (0.1, 0.3, 0.6, 1.0, or 2.0 mg/mL) or without (0.0 mg/mL) LC. Matured oocytes were fertilized in vitro with frozen–thawed spermatozoa, and zygotes were cultured in a SOF medium for 7 days. IVM rates were higher (p ≤ 0.05) in 0.3 and 0.6 mg/mL LC supplemented groups than in the control (0.0 mg/mL LC) and other LC groups. Fertilization (18 h postinsemination (pi)) and cleavage (2–16-cell stage at day 3 pi) rates were higher (p ≤ 0.05) in the 0.6 mg/mL LC group than in the control and 0.1, 1.0, and 2 mg/mL LC supplemented groups. Interestingly, 4.5% of fertilized oocytes developed to morula (day 5 pi) in the 0.6 mg/mL LC group, which was higher (p ≤ 0.05) than those developed in the 0.3 mg/mL group (1.0%). No cleaved embryos developed to morula in other groups. In conclusion, LC supplementation at 0.6 mg/mL during IVM of canine oocytes improved their maturation, fertilization, and preimplantation embryo development rates following IVF and in vitro culture (IVC). [ABSTRACT FROM AUTHOR]

Simple Summary: Alpha-2 agonists have widely been used in dogs as sedative and preanesthetic agents. These drugs have successfully been used in many different reproductive surgical procedures in dogs, including ovariectomy (OV) and ovariohysterectomy (OVH). The main problem associated with the alpha-2 agonists' use is their dose-dependent cardiovascular effects and the changes induced on organ perfusion. Nowadays, the use of Doppler ultrasonography is the most commonly used method to assess physiological and pathological organ perfusion in veterinary medicine as it is a noninvasive technique. In bitches, Doppler ultrasonography has shown its usefulness to assess placental, ovarian and uterine blood flow during normal and abnormal pregnancy. We compared the ovarian artery flow velocity by duplex Doppler ultrasound before and after sedation with two commonly used alpha-2 agonists: medetomidine and dexmedetomidine. The results showed that the administration of medetomidine or dexmedetomidine induced an important decrease in blood flow velocities in the ovarian artery. Hence, their use could be indicated in surgeries to avoid excessive bleeding of the ovarian pedicle. The aim was to evaluate if medetomidine and dexmedetomidine affected arterial ovarian blood flow in dogs. The dogs were randomly assigned to two different groups. In Group 1, medetomidine (10 µg/kg) was administered intramuscularly and, in Group 2, dexmedetomidine (5 µg/kg) was used. After a preliminary exam, arterial blood pressure (BP) was measured and a duplex Doppler ultrasonographic examination of both ovarian arteries was performed. Twenty minutes after the administration of medetomidine or dexmedetomidine, BP and ovarian Doppler ultrasonography were repeated. High quality tracings of ovarian artery flow velocity were obtained in all dogs and Doppler parameters: Peak Systolic Velocity (PSV), End Diastolic Velocity (EDV) and Resistive Index (RI) were measured before and after drug administration in the left (LO) and right (RO) ovaries. PSV and EDV values decreased significantly after drug administration (p < 0.05) compared to the non-sedated values, but no differences were found between the LO and RO (p > 0.05). The RI was not affected by drugs administration in neither of the groups studied (p > 0.05). In conclusion, the administration of medetomidine or dexmedetomidine causes a decrease in blood flow velocity in the ovarian artery and may be a good choice to avoid excessive bleeding prior surgeries in which ovariectomy. [ABSTRACT FROM AUTHOR]

Simple Summary: Shear wave elastography (SWE) is a new diagnostic method allowing for a non-invasive differential diagnosis between benign and malignant mammary tumors. Mammary tumors in dogs are often hormone-dependent, and a surgical approach is currently the most recommended method for the treatment of mammary neoplasia. However, preoperative, pharmacological therapy eliminating hormonal stimulation can sometimes be indicated to reduce the size of the tumor and facilitate subsequent surgery. The aim of this study was to evaluate the influence of the reduction in the benign tumor size observed after treatment with the use of aglepristone on the shear wave elastography results, obtained before and after therapy. We noticed that a fast and significant reduction in tumor size, as a consequence of preoperative pharmacological treatment, did not influence the diagnosis performed using the SWE technique. These results seem to indicate the accuracy and usefulness of this new diagnostic method, and confirm the hypothesis that tumor tissue density is a valuable parameter when determining the benignancy of mammary tumors in dogs. Shear wave elastography (SWE) can be useful to discriminate between malignant and benign mammary tumors. In dogs with elevated progesterone levels compared to the baseline and fast-growing tumors, treatment with the use of aglepristone allows for tumor size reduction, which facilitates surgery. This study aimed to evaluate the influence of the preoperative treatment of benign mammary tumors (BMTs), performed with the use of aglepristone, on the density of the tumor tissue measured by SWE. Twelve female dogs with diagnosed BMTs and increased levels of progesterone were treated with aglepristone (Alizine, Virbac, France) at 10 mg/kg s.c. (Subcutaneous injection). twice, with a 24 h interval. The density of the tumor was evaluated by SWE before and after the treatment. The type of tumor was evaluated by fine needle aspiration cytology before treatment, and a histopathological examination was made after surgical removal, performed after the aglepristone treatment. In all the cases, a significant reduction in the mammary tumor's size was observed following treatment, with no influence on the density of the tumor's tissue measured by SWE. Similar studies on malignant mammary tumors are warranted to verify if in these cases, density will also be a constant parameter that is not dependent on the tumor size. [ABSTRACT FROM AUTHOR]

Simple Summary: The oestrous cycle in canines is specifically more extended than that in other mammals. This implies that the oocytes do not reach maturity within the ovarian follicle but undergo final maturation in the oviducts. Besides oocyte maturation, the oviduct provides the necessary milieu for fertilization and preimplantation embryonic development. Consequently, the oviductal environment presumably changes in the postovulatory period and throughout the entire reproductive cycle to provide a suitable condition for supporting different functions. In this study, we evaluated the gene expression of different genes associated with oocyte-embryo development, such as progesterone receptor, cyclooxygenase-2, growth differentiation factor 9, and bone morphogenetic protein 15 in the canine oviductal cells at different phases of the oestrous cycle. Using quantitative PCR (qPCR) analysis in bitch oviductal cells, this study revealed the ovarian cycle's influence on the oviductal essential transcripts in the bitch. It also assessed the influence of the ovulated cumulus-oocytes complexes on the expression of GDF-9 and BMP-15 genes. Thus, the oestrous-cycle-dependent gene expression pattern of PR, COX-2, GDF-9, BMP-15 in the canine oviduct was found to execute the oviductal cell interactions necessary for the development and function of the canine reproductive tract. The gene expression in the canine oviduct, where oocyte maturation, fertilization, and early embryonic development occur, is still elusive. This study determined the oviductal expression of (PR), cyclooxygenase-2 (COX-2), growth differentiation factor 9 (GDF-9), and bone morphogenetic protein 15 (BMP-15) during the canine oestrous cycle. Samples were collected from bitches at anoestrus (9), proestrus (7), oestrus (8), and dioestrus (11), after routine ovariohysterectomy and the ovarian surface structures and plasma progesterone concentration evaluated the physiological status of each donor. The oviductal cells were isolated and pooled. Total RNA was isolated, and gene expression was assessed by qPCR followed by analysis using the t-test and ANOVA. The PR mRNA increased (P < 0.05) from the anoestrus to dioestrus with the plasma progesterone concentration (r = 0.8). COX-2 mRNA expression was low in the anoestrus and proestrus, and negligible in the oestrus, while it was around 10-fold higher (P < 0.05) in the dioestrus. The GDF-9 mRNA was expressed during all phases of the oestrous cycle and was most abundant (P < 0.05) during oestrus phase. The BMP-15 mRNA decreased (P < 0.05) in the anoestrus and proestrus phases. Thus, the transcripts were differentially expressed in a stage-dependent manner, suggesting the importance of oestrous cycle regulation for successful reproduction in dogs. [ABSTRACT FROM AUTHOR]

Simple Summary: The knowledge of physiological events associated with canine reproduction involving oocyte developmental potential is essential to increase the success of reproductive biotechnologies in this species. In mammals, the oocytes are closely surrounded by a group of cells known as the cumulus cells. Although it is not well-known how these cells interact with the oocyte to promote maturation, they may provide important answers concerning oocyte development. The competence to undergo expansion is a unique characteristic of cumulus cells which is critical for normal oocyte maturation, however, the complete expansion of these cells takes longer in canines, which has been associated with the lengthy maturation process of the oocyte. Growth Differentiation Factor 9 (GDF-9) and Bone Morphogenetic Protein 15 (BMP-15) are described as relevant players in the oocyte–cumulus cells' regulatory mechanisms. Cumulus cells express many important genes from a very early stage, therefore, we proposed to study the gene expression of GDF-9 and BMP-15 in canine cumulus cells in relation to cumulus expansion and the maturation process. We demonstrate, for the first time, that these genes are differentially expressed in canine cumulus cells throughout the estrous cycle and that this expression is related to cumulus expansion and maturity status, suggesting specific regulation. The competence to undergo expansion is a characteristic of cumulus cells (CCs). The aim was to investigate the expression of GDF-9 and BMP-15 mRNA in canine cumulus cells in relation to cumulus expansion and meiotic development over the estrous cycle. CCs were recovered from nonmatured and in vitro-matured (IVM) dog cumulus oocyte complexes (COCs), which were obtained from antral follicles at different phases of the estrous cycle. Quantitative real-time polymerase chain reaction (q-PCR) was used to evaluate the relative abundance of GDF-9 and BMP-15 transcripts from the CCs with or without signs of expansion. The results were evaluated by ANOVA and logistic regression. The maturity of the oocyte and the expansion process affected the mRNA levels in CCs. There were differences (p < 0.05) in GDF-9 and BMP-15 gene expression in CCs isolated from nonmatured COCs when comparing the reproductive phases. Lower mRNA levels (p < 0.05) were observed in anestrus and proestrus in comparison to those in estrus and diestrus. In contrast, when comparing GDF-9 mRNA levels in IVM COCs, no differences were found among the phases of the estrous cycle in expanded and nonexpanded CCs (p < 0.05). However, the highest (p < 0.05) BMP-15 gene expression in CCs that did not undergo expansion was exhibited in anestrus and the lowest (p < 0.05) expression was observed in estrus in expanded CCs. Although the stage of the estrous cycle did not affect the second metaphase (MII)rates, the expanded CCs obtained at estrus coexisted with higher percentages of MII (p < 0.05). In conclusion, the differential expression patterns of GDF-9 and BMP-15 mRNA transcripts might be related to cumulus expansion and maturation processes, suggesting specific regulation and temporal changes in their expression. [ABSTRACT FROM AUTHOR]