Ultrasound-based assessment of the expression of inflammatory markers in the rectus femoris muscle of rats.

Ultrasonographic characteristics of skeletal muscles are related to their health status and functional capacity, but they still provide limited information on muscle composition during the inflammatory process. It has been demonstrated that an alteration in muscle composition or structure can have disparate effects on different ranges of ultrasonogram pixel intensities. Therefore, monitoring specific clusters or bands of pixel intensity values could help detect echotextural changes in skeletal muscles associated with neurogenic inflammation. Here we compare two methods of ultrasonographic image analysis, namely, the echointensity (EI) segmentation approach (EI banding method) and detection of selective pixel intensity ranges correlated with the expression of inflammatory regulators using an in-house developed computer algorithm (r-Algo). This study utilized an experimental model of neurogenic inflammation in segmentally linked myotomes (i.e., rectus femoris (RF) muscle) of rats subjected to lumbar facet injury. Our results show that there were no significant differences in RF echotextural variables for different EI bands (with 50- or 25-pixel intervals) between surgery and sham-operated rats, and no significant correlations among individual EI band pixel characteristics and protein expression of inflammatory regulators studied. However, mean numerical pixel values for the pixel intensity ranges identified with the proprietary r-Algo computer program correlated with protein expression of ERK1/2 and substance P (both 86-101-pixel ranges) and CaMKII (86-103-pixel range) in RF, and were greater (p < 0.05) in surgery rats compared with their sham-operated counterparts. Our findings indicate that computer-aided identification of specific pixel intensity ranges was critical for ultrasonographic detection of changes in the expression of inflammatory mediators in neurosegmentally-linked skeletal muscles of rats after facet injury.

The effects of heat stress on intrauterine development, reproductive function, and ovarian gene expression of F1 female mice as well as gene expression of F2 embryos†.

Exposure to heat stress (HS) in utero was postulated to trigger an adaptive molecular response that can be transmitted to the next generation. Hence, this study assessed the impact of HS exposure at different stages of the gestational period of mice on the female F1 population and their offspring. Heat stress exposure (41°C and 65% relative humidity-RH) occurred during the first half (FP), the second half (SP), or the entire pregnancy (TP). A control group (C) was maintained in normothermic conditions (25°C, 45% RH) throughout the experiment. Heat stress had a significant negative effect on intrauterine development, mainly when HS exposure occurred in the first half of pregnancy (FP and TP groups). Postnatal growth of FP and TP mice was hindered until 4 weeks of age. The total number of follicles per ovary did not vary (P > 0.05) between the control and HS-exposed groups. Mean numbers of primordial follicles were lower (P < 0.05) in the sexually mature FP than those in SP and TP F1 females. However, the mean number of viable embryos after superovulation was lower (P < 0.05) in TP compared with C group. The expression of genes associated with physiological and cellular response to HS, autophagy, and apoptosis was significantly affected in the ovarian tissue of F1 females and F2 in vivo-derived blastocysts in all HS-exposed groups. In conclusion, exposure to HS during pregnancy compromised somatic development and reproductive parameters as well as altered gene expression profile that was then transmitted to the next generation of mice.

Suitability of a universal electroporation device for genome editing and production of transgenic rats.

Mammalian genome editing has utilized expensive and highly specialized electroporator devices. The "Gene Pulser XCell," a modular electroporation system for transfecting all cell types, has not been used extensively in mammalian embryo genome editing. The present experiment was undertaken to determine the usefulness of the Gene Pulser XCell for inserting the CRISPR/Cas9 system into intact zygotes in order to obtain the enhanced green fluorescent protein reporter rats (eGFP-R). An electroporation pulse response test using mCherry mRNA was performed to optimize the settings of the electroporator. Forty-five combinations of five pulse voltages (15, 25, 30, 35 and 40 V), three pulse durations (5, 10 and 25 ms), and three pulse frequencies (2, 5 and 6 pulses) applied at a constant 100-ms pulse interval and temperature of 37.5 °C were evaluated. The test revealed that the 35 V was the only voltage suitable for insertion of mCherry mRNA into intact rat zygotes and the only one that resulted in the production of embryos attaining the blastocyst stage. The incorporation of mCherry mRNA increased but the survival of the electroporated embryos declined with an increment in the number of pulses. Subsequent transfer of 1112 surviving Sprague Dawley rat embryos (after 8 h of incubating 1800 zygotes electroporated with the CRISPR/Cas9) resulted in the production of 287 offspring (25.8%). Ensuing PCR and phenotypic evaluation confirmed that twenty animals (6.96%) expressed eGFP in all body organs/tissues except for blood and blood vessels. The mortality of males and females before the attainment of puberty was 2 and 3 pups, respectively, and the final number/ratio of male to female of offspring was 9:11. All the surviving rats mated naturally and successfully transmitted the GFP transgene to their progeny. The Gene Pulser XCell total system with the settings predetermined in the present experiment can effectively be used to produce transgenic rats through the CRISPR/Cas9-mediated genome editing of zygotes.

Morphokinetic changes and apoptotic cell death in vitrified and non-vitrified in vitro-produced ovine embryos.

This article addresses morphokinetic changes and the extent of apoptosis in vitrified and non-vitrified in vitro-derived ovine blastocysts. Cumulus-oocyte complexes were collected after ovarian scarification obtain after slaughter and in vitro maturation was performed in TCM 199 medium supplemented with Earle's Salt, 10 % of FBS, and 5 µg/mL of LH/FSH at 38 °C for 24 h. After maturation, the oocytes were co-incubated with thawed ram semen (IVF) for 19 h.Embryo development was monitored with the aid of the Primo Vision Time-Lapse (TL) system. Twenty-five out of thirty-one ovine blastocysts that were vitrified using the Cryotop system at the early blastulation stage of development subsequently re-expanded. Both the vitrified (n = 25) and non-vitrified (control group: n = 28) blastocysts were examined for detection of apoptosis (TUNEL assay) and total blastomere counts at the time they attained the expanded blastocyst stage. Blastocyst formation occurred earlier in non-vitrified than in vitrified ovine embryos (147:49 ± 20:23 compared with 156:46 ± 19:24; hours:minutes post-insemination; mean ± SD; P < 0.05). The average number of blastocyst collapses was greater (2.45 ± 1.64 compared with 1.45 ± 1.64), but the number of weak contractions was less for vitrified than non-vitrified ovine blastocysts (P < 0.05). The mean number of blastomeres was greater (131.8 ± 38.6 compared with 91.5 ± 18.3; P < 0.05) while the number of TUNEL-positive cells (4.4 ± 1.6 compared with 6.3 ± 2.3) and apoptotic index (3.4 ± 1.2 % compared with 6.9 ± 2.6 %) were less (P < 0.05) in non-vitrified compared with vitrified blastocysts. Vitrification of ovine embryos was associated with a delayed blastocyst formation, greater numbers of apoptotic cells, significant reduction in the number of blastomeres, and higher/lower incidence of blastocyst collapse/weak contractions.

Ultrasonographic assessment of skeletal muscles after experimentally induced neurogenic inflammation (facet injury) in rats.

This study set out to examine ultrasonographic attributes of non-neurosegmentally (pectoral-forelimb) and neurosegmentally linked (hindlimb) myotomes in an experimental model that leads to neurogenic inflammation in segmentally linked myotomes, and to evaluate quantitative correlations among ultrasonographic attributes of the muscles, relative content of various inflammatory mediators, and nociceptive thresholds (hot and mechanical) in rats. Twelve male Wistar Kyoto rats were randomly divided into two equinumerous groups: surgery group, in which the left lumbar (L4-L6) facet joints were compressed for 3 min with modified Kelly forceps under general anesthesia, and sham-operated rats. All ultrasonograms were obtained with the Vevo 2100 Visual Sonic scanner connected to a 24-MHz transducer at four different time points: pre-surgery and 7, 14, and 21 days after surgical procedures. Digital ultrasonographic images of quadriceps femoris, hamstring, and pectoral-brachial muscle groups were analyzed using a polygonal meter region of interest placed on the largest cross-sectional area of the muscles displayed in Image ProPlus® analytical software to compute numerical pixel values and pixel heterogeneity (standard deviation of mean pixel values). On day 21, pain behavior tests (hot plate and von Frey) were performed and then all animals were euthanized. Protein expression of inflammatory mediators in biceps brachii and rectus femoris muscles was measured by Western blot. The most prominent differences in muscle echotextural attributes between the two subsets of rats occurred 14 days post-surgery in pectoral-brachial and quadriceps femoris muscles. The expression of calcitonin-gene-related peptide was directly related to both echotextural variables only in biceps brachii (pixel intensity: r = 0.65, P = 0.02; and heterogeneity: r = 0.66, P = 0.02, respectively). Our findings have revealed the occurrence of echotextural changes in skeletal muscles of rats during myositis; however, the accumulation of inflammatory mediators and the outcomes of sensory tests did not relate to the changes in first-order echotextural characteristics of affected hindlimb muscles.

Morphokinetic changes in vitrified and non-vitrified in vitro-derived ovine embryos.

The present experiment employed time-lapse (TL) imaging to assess the effects of vitrification on the development of ovine blastocysts and to see if the timing of blastocyst formation and expansion was correlated with the numbers of embryo- and trophoblasts determined through differential staining of the expanded blastocysts. Ovaries were obtained after slaughter from cycling (October-March) Polish Longwool ewes aged 1-3 years and cumulus-oocyte complexes were collected by ovarian scarification. In vitro maturation was performed in TCM 199 medium supplemented with Earle's Salt, 10% of FBS, and 5 µg/mL of LH/FSH at 38 °C for 24 h. After maturation, the oocytes were incubated with thawed ram semen (IVF) for 19 h and all presumptive zygotes were transferred to a 16-well dish containing Cult medium for monitoring with the Primo Vision TL system. A portion of ovine embryos were vitrified (Cryotop system) at the early cavitation stage and TL observations of warmed (n = 30) and non-vitrified (n = 32) blastocysts continued until the attainment of the expanded blastocyst stage, at which point they were differentially stained with bisbenzimide and propidium iodide for microscopic enumeration of embryoblasts (inner cell mass blastomeres) and trophoblast-cells. There were no statistically significant differences in the timing of blastulation (tB) and formation of expanded blastocysts (tEB) between vitrified and non-vitrified ovine embryos, but non-vitrified blastocysts exceeded (P < 0.05) their vitrified and warmed counterparts in the mean number of embryo- and trophoblasts. In addition, the number of trophoblasts was negatively and moderately correlated with tB and tEB, for both vitrified and non-vitrified embryos. It can be concluded that even though vitrification of ovine embryos is associated with a significant reduction in the number of blastomeres, the rate of blastocyst development remains closely linked to the numbers of trophoblastic cells.

The roles and expression of HOXA/Hoxa10 gene: A prospective marker of mammalian female fertility?

This review addresses the influence of homebox A10/a10 (HOXA/Hoxa10) gene on reproductive tract anatomy and functional fertility in mammalian species, and discusses major endocrine and environmental regulators of HOXA/Hoxa10 expression. Female reproductive efficiency or success is a function of several factors including the ovulation and fertilization rate, and uterine receptivity. A family of HOX/Hox genes establishes the segmental identity of the reproductive tract during embryogenesis and retains its physiological plasticity in sexually mature animals and humans. In particular, the HOXA/Hoxa10 gene is an intrinsic component of implantation, decidualization, and immunomodulation in the adult uterus. It was, therefore, suggested that knowledge of HOXA/Hoxa10 regulation might be essential in navigating molecular mechanisms with the aim of enhancing female reproductive potential. However, a recent study in pigs revealed a lack of associations between endometrial HOXA10 expression and reproductive tract morphology, and very poor correlations with sows' fertility metrics. Retinoic acid mainly regulates 3' HOX/Hox paralogs but may also modify the expression of downstream HOX/Hox genes, including HOXA/Hoxa10. Sex steroids directly regulate HOXA/Hoxa10 expression. The vitamin D receptor pathway modulates HOXA/Hoxa10 expression in the adult reproductive tract. Lastly, endocrine disruptors such as diethylstilbestrol, methoxychlor, bisphenol A, and isoflavones were shown to alter HOXA/Hoxa10 expression, thus affecting reproductive competence of the female.

Correlates of reproductive tract anatomy and uterine histomorphometrics with fertility in swine.

Economic potential of the swine industry hinges upon the reproductive performance of sows, which may be enhanced by improving uterine capacity, a component trait of litter size and piglet productivity. Previous attempts at characterizing morphological traits indicative of high uterine volume have not been completely successful, resulting in the continued need for a reliable method of predicting reproductive value to improve production efficiency of the sow. Hence, the main objective of this study was to scrutinize macro- and micro-morphology of the sow's reproductive tract for quantitative correlations with fertility indices. Reproductive records from Polish Landrace × Polish Large White sows were used to examine the associations between fertility and ovarian/uterine morphology (n = 34) or uterine histomorphometry (n = 10). Several measures related to the ovary, including right and left ovarian weight (r = 0.50, p = 0.005 and r = 0.49, p = 0.006, respectively), were positively correlated with the litter size, while left ovarian number of corpora lutea (r = -0.38, p = 0.04) was negatively correlated with the mean litter size. Analysis of histomorphological characteristics of the uterine wall collected during the luteal phase of the estrous cycle revealed correlations between mean litter size and myometrial vascular content (r = 0.75, p = 0.03), the proportion of myometrial stroma (r = -0.68, p = 0.03), and the variability of endometrial thickness (r = -0.72, p = 0.02) in sows. Eight ovarian, vaginal and uterine characteristics were significantly correlated with mean lifetime numbers of live born and stillborn piglets/litter or the last litter size before slaughter. In conclusion, several anatomical and histomorphological metrics that relate to reproductive performance of swine may be used to inform production protocols and as a tool for selection of elite breeding sows, warranting future research into non-invasive or minimally invasive techniques for obtaining such measures.

Correlations among Ultrasonographic, Physicochemical and Sensory Characteristics of Pectoralis Major Muscles in Turkeys Reared in a Sustainable Farming System.

This study set out to examine associations among echotextural, physicochemical and sensory attributes of the pectoralis major muscles in 17-week-old organic turkeys (B.U.T. Big-6) varying in the amount of wheat and oat grain in daily feed rations (Group C: complete feed only; Group Exp1: 5-30% of wheat and 0-20% of oat; and Group Exp2: 5-50% of wheat and 0-50% of oat; n = 15 turkeys/group). Digital ultrasonograms of the left pectoral muscle in four different planes (longitudinal-L, transverse-T, and two oblique planes-O1 and O2) were obtained with a 5.0-MHz linear-array transducer just before slaughter. Mean numerical pixel intensity (MPI) and pixel heterogeneity (MPH) of the muscle parenchyma were computed using the ImageProPlus® analytical software. Ten significant correlations between echotextural attributes and various meat characteristics were recorded in Group C, one in Group Exp1, and eight in Group Exp2. When data were pooled for all birds studied, there were twelve significant correlations (p < 0.05); all but one correlation (between MPH and moisture) were for physical and sensory characteristics of meat samples. Computer-assisted analysis is a potential method to determine moisture as well as physical (e.g., coloration) and sensory (e.g., aroma) characteristics of pectoralis major muscles in organic turkeys.

Timing of cleavage divisions determined with time-lapse imaging is linked to blastocyst formation rates and quality of in vitro-produced ovine embryos.

Time-lapse (TL) imaging provides a practical and safe tool to constantly monitor the development of in vitro-derived embryos. TL may help develop novel methods of predicting the timing of embryo cleavage that will lead to optimizing blastocyst cryopreservation or transfer. The primary objective of the present study was to employ TL imaging to examine associations among the division kinetics of ovine embryos, their quality and rates of development to the blastocyst stage. Oocytes were collected by ovary scarification from 78 Longwool ewes slaughtered in the breeding season (November-March). Cumulus oocyte complexes (COCs) were matured for 24 h in TCM 199 media containing 0.1 IU/mL LH/FSH and 10% FBS. In-vitro fertilization was carried out by co-incubation of semen and COCs for 19 h. Presumptive zygotes were placed in microwells, in droplets of Cult medium (Gynemed, Lensahn, Germany). Digital images of developing embryos were captured every 10 min by Primo Vision TL system (EVO+; Vitrolife, Göteburg, Sweden). The following time intervals were recorded: from IVF to the attainment of two-cell (t2), three-cells (t3) or four-cell (t4) stage, to morula detection (tM), blastulation (tSB) and blastocyst formation (tB). Lastly, the duration of the second cell cycle (cc2; t3-t2) and complete synchronous cell division (s2; t4-t3) were calculated, and the incidence of developmental anomalies noted. Out of 147 embryos selected for TL observations, 55 (37.4%) developed to the blastocyst stage (normally developing embryos, NE) and 92 (62.6%) failed to reach the blastocyst stage (arrested embryos, AE; P < 0.05). Mean t2, tM, s2 and cc2 were all less (P ≤ 0.02) in NE compared with AE. Approximately 61.9% of embryos exhibited developmental anomalies (35.5% in the NE group and 78.2% in the AE group; P < 0.05) and AE exceeded (P < 0.05) NE in the proportion of FRG (blastomeric fragmentation), IRR (blastomeres of irregular size after cleavage), DC (direct cleavage) and MA (multi-morphological aberrations). Of all NE, 63.6% were classified as good quality and 36.4% as poor quality blastocysts (P < 0.05). Good quality ovine blastocysts attained t2, t3, t4, tSB and tB stages earlier (P ≤ 0.03) than poor quality blastocysts and none of the poor quality blastocysts was seen to hatch. To recapitulate, the present results indicate that the kinetics of early ovine embryo development are significant predictors of their potential to develop to the blastocyst stage and the markers of blastocyst quality. Time-lapse imaging may serve as a useful technique for predicting the outcome and enhancing efficacy of in vitro embryo production in sheep.

Associations between Mammary Gland Echotexture and Milk Composition in Cows.

Thirty clinically healthy Holstein-Friesian cows underwent twice daily machine milking and ultrasonographic examinations of the udder just prior to and after milking. Digital ultrasonographic images of each udder quarter were subjected to computer-assisted echotextural analyses to obtain mean numerical pixel values (NPVs) and pixel heterogeneity (PSD) of the mammary gland parenchyma. The average milk yield and pH were higher (p < 0.05) in the morning, whereas crude fat, total solids, solids non-fat and citric acid content were higher (p < 0.05) during the evening milking period. Mean NPVs and PSDs of the mammary gland parenchyma were greater (p < 0.05) after than before milking. There were significant correlations among echotextural characteristics of the udder and protein percentage, lactose content and freezing point depression determined in the milk samples collected in the morning and crude protein, casein, lactose and solids non-fat in the evening. Our results can be interpreted to suggest that computerized analysis of the mammary gland ultrasonograms has the makings of a technique for estimating non-fat milk constituents in cows. However, future validating studies are necessary before this method can be employed in commercial settings and research. Moreover, significant inter-quarter differences in udder echogenicity may necessitate further echotextural studies of separate quarters.

Quantitative Echotextural Attributes of Pectoralis Major Muscles in Broiler Chickens: Physicochemical Correlates and Effects of Dietary Fat Source.

This study examined the relationships among physicochemical properties and ultrasonographic image attributes of pectoralis major muscles in broiler chickens. Forty male Ross 308 chicks were randomly assigned to four equinumerous fat-supplementation groups (Group SO: soybean oil; Group FO: flax oil; Group SO + FO: soybean oil + flax oil; and Group BF: beef fat). Ultrasonograms of birds' pectoral muscles were obtained just before slaughter at 6 weeks of age and were subjected to digital image analyses to determine the mean pixel intensity (MPI) and pixel heterogeneity values (standard deviation of numerical pixel values; MPH). A total of 2, 4, 2, and 6 significant correlations were recorded in Groups SO, FO, SO + FO, and BF, respectively; there were no correlations with the chemical composition of the muscles in Groups SO and SO + FO. The strongest correlations were found between muscle lightness (L*) and MPH in Group BF (physical characteristic; r = -0.82, p = 0.003), and between crude fat/protein content and MPI/MPH of pectoral the major muscles in Groups FO/BF (chemical characteristics; r = 0.72, p = 0.02). There exists a potential application of ultrasonographic imaging and computerized image analysis for predicting certain physicochemical properties of pectoralis major muscles in broiler chickens.

Changes in testicular size, echotexture, and arterial blood flow associated with the attainment of puberty in Dorper rams raised in a subtropical climate.

There is a paucity of information on the relationships of testicular morphology, echotextural attributes, and blood flow dynamics with pubertal development of rams raised in a subtropical climate. Forty-five Dorper rams (24 rams aged 8-11 months and 21 rams aged 12-24 months) were examined using a portable ultrasound scanner connected to a 7.5-MHz transducer. Computer-assisted analyses of testicular ultrasonograms utilized commercially available Image ProPlus® analytical software. Spectral Doppler scans of testicular arteries were performed immediately after scrotal (B-mode) ultrasonography to determine peak systolic velocity (PSV), end-diastolic velocity (EDV), resistive index (RI = [PSV-EDV]/PSV), and pulsatility index (PI = [SPV-EDV]/mean velocity) of the blood vessels. The length of the testes (9.7 ± 0.3 compared with 9.0 ± 0.2 cm) and scrotal circumference (33.3 ± 0.5 compared with 31.8 ± 0.4 cm) were greater (p < 0.05) but testicular depth (4.5 ± 0.1  compared with 4.9 ± 0.08 cm) was less (p < 0.05) in sexually mature compared with peripubertal rams. [Corrections added on 9 Jan 2019 after initial online publication: The testicular size values in the sentence were corrected.] There were no differences (p > 0.05) between the two age groups of Dorper rams in blood flow indices of testicular arteries. Mean numerical pixel values (100.5 ± 4.1 compared with 89.2 ± 4.8) and pixel heterogeneity (25.6 ± 0.6 compared with 23.6 ± 0.5) of testicular parenchyma were greater (p < 0.05) in peripubertal than in postpubertal rams. Semen volume was negatively correlated with PI of testicular arteries (r = -0.57, p = 0.04). In summary, the attainment of sexual maturity in the rams of the present study was associated with significant changes in testicular length and depth, scrotal circumference, and parenchymal echogenicity/hetrogeneity but not in testicular volume and blood perfusion rates. Testicular artery PI can be used to predict the volume of ejaculate in rams.

Effects of co-incubation with conspecific ampulla oviductal epithelial cells and media composition on cryotolerance and developmental competence of in vitro matured sheep oocytes.

Developmental potential of cryopreserved in vitro matured oocytes is very low in nearly all mammalian species studied to date. Despite relatively high cleavage rates, the vitrified/warmed metaphase II oocytes have a decreased rate of blastocyst formation, which can be attributed to the elevated cytoplasmic lipid content and lipid droplet fragmentation. Secretory products of ampulla oviductal epithelial cells (AECs) at the periovulatory stage of the ovarian cycle enhance the viability of in vitro matured oocytes. The present study was undertaken to determine if co-culture of cumulus-oophorus complexes (COCs) with conspecific AECs or reducing the lipid content of in vitro matured ovine oocytes would improve their cryotolerance and ensuing developmental competence. Ovine COCs aspirated from the slaughterhouse ovaries were matured in the following media or culture conditions: TCM199 + FBS + AECs (T1); TCM199 + FBS (T2); TCM199 + BSA (T3); TCM199 + 0.6 mg/mL of l-carnitine (T4); TCM199+ l-carnitine + FBS (T5), or TCM199 only (Ctr). Subsequently, the oocytes were vitrified and used for in vitro fertilization (IVF). The lowest degree of zona pellucida (ZP) hardening following vitrification of in vitro matured sheep oocytes was observed in T1 and T5 (P < 0.05). Cleavage, blastocyst formation and ensuing development (i.e., total cell numbers) as well as blastocyst hatching rates were all greater (P < 0.05) in T1 compared with the remaining groups; in vitro matured COCs in T4 and Ctr did not develop beyond the cleavage stage. The inner cell mass: trophectoderm cell ratio in T1 (1:3.29) was significantly greater compared with T2 (1:3.39), T3 (1:3.40) and T5 (1:3.44). The present results indicate that the ovine COCs/AECs co-culture system had the most positive influence on cryotolerance, ZP hardening, and developmental competence of in vitro matured oocytes.

Non-surgical embryo transfer in goats and sheep: the Brazilian experience.

Brazil has presented tremendous progress in non-surgical embryo transfer (NSET) in sheep and goats. New instruments and techniques for non-surgical embryo recovery (NSER) and NSET in small ruminants were implemented. Recent improvements include refinement of the protocols for cervical relaxation combining oestradiol-oxytocin-cloprostenol treatment at specific times before NSER in sheep; recipient goats do not require any hormonal drugs to induce cervical dilation and direct embryo transfer by the cervical route yields excellent results. Transrectal ovarian ultrasonography (B-mode but especially colour Doppler) have proven to be accurate methods to localise and enumerate corpora lutea and luteinised unovulated follicles in recipient and donor does and ewes. An array of new criteria for selecting superior animals for NSER and NSET (e.g. cervical mapping) have been developed by Brazilian researchers. Extensive studies on both technologies were initially conducted in commercial breeds of goats and sheep but have been gradually extended to some native breeds of sheep (germplasm conservation) and dairy goat operations. It is speculated that, in future, NSER and NSET may become methods of choice for caprine and ovine embryo recovery and transfer in Brazil, and then globally. Due primarily to the efficiency of NSET in goats, a novel interspecies (e.g. bovine) IVP method may soon be developed on a large scale. The Brazilian experience is an invaluable source of information and know-how promoting the replacement of conventional surgical assisted reproductive technologies with non-surgical procedures and hence supporting the rapid development of the embryo transfer industry in small ruminants.

Prospective ultrasonographic and endocrine predictors of spermatogenic onset in ram lambs.

The objective of this study was to examine testicular ultrasonographic characteristics and endocrine profiles in prepubescent ram lambs for correlations with the age at first detection of elongated spermatids (ESt age). Bi-weekly ultrasound examinations and weekly testicular biopsies began at 10 weeks of age or at the time that testicular volume reached 15cm3, and continued until 1-2 weeks after Est's were first detected by histological examination of testicular biopsies in twenty-two spring-born Rideau Arcott×Polled Dorset lambs. Computer-assisted analysis of testicular ultrasonograms was performed using commercially available image analytical software. Blood samples were drawn before each ultrasonographic examination and were used for measurements of free triiodothyronine (fT3) and thyroxine (fT4), and follicle-stimulating hormone (FSH) concentrations. The mean (±SEM) age at first detection of ESts was 15.9±0.5 weeks. Testicular volumes recorded between 10 and 12 weeks of age correlated inversely with the ESt age (r=-0.44 to -0.50, P≤0.05). Statistically significant correlations were recorded between the ESt age and numerical pixel values of testicular parenchyma at 10 (r=-0.48, P=0.05) and 15 (r=0.52, P=0.05) weeks of age, and between the ESt age and testicular pixel heterogeneity in ram lambs aged 14.5 weeks (r=0.60, P=0.007). Lastly, circulating FSH concentrations at 10 weeks (r=-0.43, P=0.05), serum fT3 concentrations at 13 weeks (r=0.44, P=0.04) and fT4 concentrations at 11.5 weeks of age (r=0.48, P=0.03) were all correlated with the ESt age. The present results show that testicular volume has the most stable relationship with pubertal onset; however, testicular echotexture as well as circulating concentrations of FSH and free fractions of thyroid hormones at specific ages may be indicative of more intricate developmental events heralding puberty.

The effect of conspecific ampulla oviductal epithelial cells during in vitro maturation on oocyte developmental competence and maturation-promoting factor (MPF) activity in sheep.

The acquisition of fertilization ability by oocytes is one of the prerequisites for successful in vitro embryo production. In the present study, we examined the influence of conspecific ampulla oviductal epithelial cells incubated with cumulus-oocyte complexes (COCs) throughout the IVM phase on the developmental competence and maturation-promoting factor (MPF) activity of sheep oocytes. There were six experimental groups in this study, namely four groups with and two groups without oviductal epithelial cells added to IVM media: adult COCs matured in vitro with the ampulla oviductal epithelial cells obtained from adult (AAE; G1) or prepubertal donors (prepubertal sheep ampulla oviductal epithelial cells [PAE]; G4), COCs obtained from prepubertal animals cocultured with AAE (G2) or PAE (G3), and adult (C1) and prepubertal sheep COCs (C2) matured without oviductal epithelial cells. Coincubation of oocytes retrieved from both adult and sexually immature donors with AAE (G1 and G2) resulted in significantly improved rates of metaphase-II (M-II) attainment (G1: 85.1 ± 2.0 and G2: 40.2 ± 1.3) and blastocyst formation (G1: 42.2 ± 1.1 and G2: 21.2 ± 1.0) as well as blastocyst development (total cell count; G1: 130.3 ± 7.8, G2: 70.2 ± 3.5) compared with their respective controls (C1: 94.3 ± 4.1 and C2: 49.7 ± 2.0). Prior to IVM, the activity of MPF was greater (P < 0.05) for oocytes obtained from ewes (G1, G4, and C1) compared with those from ewe lambs (G2, G3, and C2). The greatest increment in MPF activity was recorded in G2 (MPF activity before IVM/MPF activity after IVM = 3.62) followed by C2 and G3 (2.22 and 2.20, respectively), and then all remaining groups of oocytes (C1: 1.89, G1: 1.87, and G4: 1.86). In summary, coincubation with AAE during the 24-hour IVM had a positive impact on ovine oocyte competence and ensuing in vitro embryo production efficiency. A significant increase in MPF activity following IVM of G2 oocytes could be responsible, at least partly, for the improved rate of blastocyst formation after IVF of prepubertal sheep oocytes.

Retinoic acid treatment alters germ cell heterogeneity and testicular echotexture in prepubescent ram lambs.

Testicular echotextural attributes are closely associated with spermatogenic development; however, precise characterisation of specific germ cell types is difficult due to tremendous germ cell heterogeneity. Recently, retinoic acid (RA) administration in neonatal mice was found to induce highly synchronised spermatogenesis as adults. A RA-treatment protocol was tested in 17 ram lambs treated with or without RA at 8 weeks of age, with scrotal ultrasonography and blood samples collected until castration 24h or 2.5 weeks later. At 8.2 weeks of age, the nuclear:seminiferous tubule (ST) area was higher in the treated compared with the control group. Serum testosterone concentrations and numerical pixel values (NPVs) of the testicular parenchyma reached a peak at 9 weeks of age in both groups of ram lambs studied. At 10.5 weeks of age, the percentage of ST cross-sections with different germ cells as the most mature germ cell type was lower and the inter-tubular heterogeneity and NPVs were also lower in the treated compared with the control animals. RA manipulation of spermatogenesis in prepubertal ram lambs may provide a suitable model for further investigation of the echotextural characteristics of specific germ cell types and critical developmental events.

Intrinsic determinants and predictors of superovulatory yields in sheep: Circulating concentrations of reproductive hormones, ovarian status, and antral follicular blood flow.

Hormonal ovarian superstimulation has contributed to small ruminant reproduction around the world, impacting genetic improvement and zoosanitary programs, contributing to the conservation of endangered species, and supporting other related biotechnologies. Advanced knowledge surrounding the superovulatory treatments in sheep has resulted in enhanced control of influencing factors and improved the protocols currently used. However, in spite of minimization of some adverse factors, superovulatory responses in ewes still remain variable, preventing the more widespread use of superovulation in commercial embryo transfer programs and reproductive research in this species. Recent evidence demonstrates that changes in antral follicular populations and blood supply, and circulating concentrations of certain reproductive hormones determined at the specific time points just before or during the superovulatory treatment are associated with superovulation success in ewes. This review attempts to compile the data from available literature to identify ovarian and hormonal determinants of the superovulatory outcome in ewes, which can be used to substantially improve the existing protocols and to reduce the extra cost and unnecessary stress imposed on poorly responding animals. An overview of most commonly used and some recently developed, FSH-based ovarian stimulation protocols is given at the outset to highlight variation in the frequency and timing of gonadotropin injections, estrus synchronization methods, and follicular wave synchronization and/or ovulation induction techniques during the superovulatory treatments in ewes.

Assessing the usefulness of prostaglandin E2 (Cervidil) for transcervical artificial insemination in ewes.

The underlying theme of this study involved the evaluation of the dilatory effects of prostaglandin E2 on the ovine cervix and thus the assessment of its potential applicability to transcervical artificial insemination (TCAI) in ewes. A novel method of prostaglandin E2 administration (controlled slow-release vaginal inserts) was examined, and the practical implications of this approach including cervical penetrability and posttreatment pregnancy rates were evaluated. The Guelph method of TCAI was performed during the seasonal anestrus (n = 40) and the breeding season (n = 40) on multiparous Rideau Arcott × Polled Dorset ewes, with or without the pretreatment with Cervidil (for a duration of 12 hours or 24 hours before TCAI). Cervical penetration rates averaged 82.5% (66 of 80), and they varied neither (P > 0.05) between the two seasons nor between Cervidil-treated ewes and their respective controls. Cervidil priming significantly reduced the total time required for TCAI during the breeding season in comparison with controls (54 vs. 98 seconds), especially after the 24-hour exposure (38 vs. 108 seconds). The time taken to traverse the uterine cervix was negatively correlated (P < 0.05) with the breed (percentage of Rideau Arcott genotype) and lifetime lamb production in seasonally anestrous ewes. Four out of 36 (11%) successfully penetrated ewes in the breeding season (three ewes allocated to the 12-hour control group and one ewe that had received Cervidil for 12 hours) became pregnant and carried the lambs to term. Vaginal mucus impedance at TCAI was significantly and positively correlated with the total time required to complete the procedure in cyclic ewes, and the negative correlation between vaginal mucus impedance and total time values at the time of controlled intravaginal drug release device removal approached to significance in anestrous ewes. The present results indicate a moderate benefit of using Cervidil for inducing cervical dilation before TCAI in ewes, mainly in the breeding season. The specific reason(s) for impaired fertility after the TCAI using frozen-thawed ram semen remains to be elucidated.