Transcervical uterine flushing and embryo transfer in sheep: Morphophysiological basis for approaches currently used, major challenges, potential improvements, and new directions (alas, including some old ideas).

At present, the success of non-surgical embryo recovery (NSER) and transfer (NSET) hinges upon the cervical passage of catheters, but penetration of the uterine cervix in ewes is problematic due to its anatomical structure (i.e., long and narrow cervical lumen with misaligned folds and rings). It is a major obstacle limiting the widespread application of NSER and NSET in sheep. While initial attempts to traverse the uterine cervix focused on adapting or re-designing insemination catheters, more recent studies demonstrated that cervical relaxation protocols were instrumental for transcervical penetration in the ewe. An application of such protocols more than tripled cervical penetration rates (currently at 90-95 %) in sheep of different breeds (e.g., Dorper, Lacaune, Santa Inês, crossbred, and indigenous Brazilian breeds) and ages/parity. There is now sufficient evidence to suggest that even repeatedly performed cervical passages do not adversely affect overall health and reproductive function of ewes. Despite these improvements, appropriate selection of donors and recipients remains one of the most important requirements for maintaining high success rates of NSER and NSET, respectively. Non-surgical ovine embryo recovery has gradually become a commercially viable method as even though the procedure still cannot be performed by untrained individuals, it is inexpensive, yields satisfactory results, and complies with current public expectations of animal welfare standards. This article reviews critical morphophysiological aspects of transcervical embryo flushing and transfer, and the prospect of both techniques to replace surgical methods for multiple ovulation and embryo transfer (MOET) programs in sheep. We have also discussed some potential pharmacological and technical developments in the field of non-invasive embryo recovery and deposition.

Luteal Function, Biometrics, and Echotextural Attributes in Santa Inês Ewes Superovulated with Different Total Doses of Porcine Follicle-Stimulating Hormone.

Premature regression of corpora lutea (PRCL) may adversely affect the outcome of hormonal ovarian superstimulation in small ruminants, and the total dose of exogenous gonadotropins used may be one of the causes of this condition. There were two major objectives of the present study: (1) to evaluate the effects of different superovulatory doses of porcine follicle-stimulating hormone (pFSH) on the biometry, blood perfusion (Doppler), and echotextural characteristics of luteal structures; and, (2) to determine the usefulness of biometric, vascular, and echotextural luteal variables, as well as measurements of circulating progesterone (P4) concentrations for early detection of PRCL in superovulated Santa Inês ewes. Twenty-seven Santa Inês ewes received an intravaginal P4-releasing device (CIDR) from Days 0 to 8 (Day 0 = random day of the anovulatory period). An IM injection of d-cloprostenol (37.5 µg) was given at the time of the CIDR insertion and withdrawal. On Day 6, all the ewes received 300 IU of eCG IM and were divided into three treatment groups (each n = 9): G100 (100 mg); G133 (133 mg); and G200 (200 mg of pFSH) administered IM every 12 h in eight injections. Transrectal ovarian ultrasonography and jugular blood sampling for serum P4 measurements were performed on Days 11 to 15. On the day of embryo recovery (Day 15), all the ewes underwent diagnostic videolaparoscopy and were classified, based on their luteal characteristics, into three response groups: nCL (ewes with normal CL only); rCL (ewes with regressing CL only); and ewes with both nCL and rCL following the superovulatory regimen. Our present results indicate that the total pFSH doses of 100 mg and 200 mg result in similar ovulatory responses and luteal function/biometrics, although the percentage of donor ewes with nCL was greater (p < 0.05) for G100 compared with the G200 animals. An application of 133 mg of pFSH was associated with diminished luteogenesis. Lastly, circulating P4 concentrations, ultrasonographic estimates of total luteal area, and CL pixel heterogeneity (standard deviation of numerical pixel values) are promising markers of luteal inadequacy in superovulated ewes.

Luteal function in cyclic goats treated with human chorionic gonadotropin administered by intramuscular or intravaginal routes at the time of artificial insemination.

Human chorionic gonadotropin (hCG) has been used to improve goats reproductive efficiency. This study aimed to (i) evaluate if hCG administered by the intramuscular (i.m.) or intravaginal (i.vag.) route can be detected by a rapid ß-hCG test in blood plasma samples and (ii) document ovarian effects of hCG administered by both routes at the time of artificial insemination (AI) performed 60 h after oestrus synchronization in goats. Twenty-two Alpine goats received two i.m. injections of 30 µg of d-cloprostenol (Prolise®, Tecnopec, São Paulo, Brazil) 7.5 days apart. One day after the onset of oestrus (at the time of AI), the goats were randomly allocated to one of the three groups that received: control (n = 7): 0.3 ml of saline solution intravaginally; hCGi.m. (n = 7): 300 IU of hCG (Vetecor®; Hertape-Calier, São Paulo, Brazil) i.m. and hCGi.vag. (n = 8): 300 IU of hCG deposited intravaginally. Blood samples were drawn at -1, 3, 6, 9 and 24 h after as well as on days 3, 7, 10, 13, 17 and 21 after hCG treatment/AI. All animals tested negative for hCG (ECO Diagnóstica, Corinto, Brazil) at -1 h, and all control animals tested negative throughout the entire blood collection period. All hCGi.m. animals tested positive from 3 h until D3 post-AI but only 50% of hCGi.vag. goats tested positive during the present study. In all animals studied, mean circulating P4 concentrations increased (p < .05) from D3 to D7 after AI and then declined (p < .05) from D10 to D17 in control and hCGi.m. groups and from D17 to D21 in the hCGi.vag. group. Total cross-sectional luteal area (CLA), mean colour Doppler area (DA), DA/CLA, mean high-velocity Doppler area and HVDA/CLA all declined (p < .05) by D17-D21 in all animals studied. In summary: (i) human chorionic gonadotropin could consistently be detected in blood samples using the rapid ß-hCG test only in the hCGi.m. group; and (ii) there were no significant differences in the mean pregnancy rate, circulating P4 concentrations and various luteal parameters studied among Control, hCGi.m. and hCGi.vag. dose.

Relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos to their developmental competence in vitro: a preliminary assessment using time-lapse imaging.

The assessment of morphology and digital image opacity may provide valuable information on the present embryo quality. Time-lapse imaging has been employed in research to establish a means of monitoring the dynamic nature of preimplantation embryo development. The aim of present study was to use time-lapse imaging for assessing various prospective morphometric and phototextural markers of the developmental potential of in vitro-derived ovine embryos. Oocytes were obtained by scarification of ovaries from nine Polish Longwool ewes. After in vitro maturation (IVM) and fertilization (IVF) of oocytes with fresh ram semen, the development of embryos to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Commercially available Image-Pro® Plus software was used to measure zona pellucida thickness, embryo diameter, total area of the perivitelline space, cellular grey-scale pixel intensity and cellular pixel heterogeneity. Statistical assessment of all attributes was done at various time points during embryo development (i.e., presumptive zygote stage: t(0); first cleavage detected at t(2) or t(3); and second cleavage detected at t(4) or t(6)). Out of thirty-seven zygotes analyzed in this study, five did not divide, 26 arrested before and six developed to the blastocyst stage. Our present results indicate that most parameters analyzed did not differ among embryos varying in their developmental fate except for the perivitelline space area that was greater (P<0.05) for non-dividing zygotes than future blastocysts at the presumptive zygote stage (4040±1850 vs. 857±262 µm2, respectively; means±SEM). Consequently, the measurement of perivitelline space at t(0) can potentially be used to prognosticate developmental potential of in vitro-produced ovine embryos albeit further confirmational studies are needed.

Scrutinizing Pork Price Volatility in the European Union over the Last Decade.

The aim of this study was to analyze the factors that can influence pork prices, particularly the effects of various types of fluctuations on the volatility of pork prices in the European Union as a whole market and individual EU countries. The research material consisted of monthly time series of pork prices collected from 2009 to 2020. These data originated from the Integrated System of Agricultural Information coordinated by the Polish Ministry of Agriculture. Information on global pork production volumes is from the Food and Agriculture Organization Statistics (FAOSTAT) database. Time series of prices were described by the multiplicative model, and seasonal breakdown was performed using the Census X-11 method. The separation of the cyclical component of the trend was performed using the Hodrick-Prescott filter. In 2019, pork production in the European Union totaled 23,954 thousand tonnes, which accounted for 21.8% of global pork production. The largest producers were Germany, Spain, and France, supplying more than half of the pork to the entire European Union market. Pork prices in the EU, averaged over the 2009-2020 period were Euro (EUR) 154.63/100 kg. The highest prices for pork were recorded in Malta, Cyprus, Bulgaria, and Greece, whereas the lowest prices in Belgium, the Netherlands, Denmark, and France. The breakdown of the time series for pork prices confirmed that, in the period from 2009 to 2020, pork prices exhibited considerable fluctuations of both a long-term and medium-term nature as well as short-term seasonal and irregular fluctuations. Prices were higher than average in summer (with a peak in June-August) and lower in winter (January-March). Overall, the proportions of different types of changes in pork prices were as follows: random changes-7.9%, seasonal changes-36.6%, and cyclical changes-55.5%.

Study of the factors affecting embryo yields and quality in superovulated Morada Nova ewes that underwent non-surgical uterine flushing.

The present study compared the outcomes of in vivo embryo production in Morada Nova ewes subjected to either 9-day (G-9SOV , n = 21) or 12-day (G-12SOV , n = 21) progesterone (P4 )-based estruses synchronization protocol coupled with superovulatory treatment with decreasing doses of porcine follicle-stimulating hormone (133 mg of pFSH given over 3 days). Non-surgical embryo recovery (NSER) was performed 6-7 days after the onset of oestrus. Total antral follicle count doubled from the first to the sixth pFSH dose in both groups (p < .05). Oestrus responses did not vary between the two groups of animals (95.2%). Corpora lutea (CL) were detected in 85.0% and 60.0% of ewes that previously manifested oestrus behaviour in G-9SOV and G-12SOV respectively. NSER was successfully completed in 86.2% of ewes that had CL (p > .05). The mean number of CL per ewe/successfully flushed donor ewe was greater (p < .05) in G-12SOV (12.3 ± 1.7/12.1 ± 1.9) than in G-9SOV (7.9 ± 1.4/8.2 ± 1.6). Mean numbers of retrieved blastocysts and viable embryos were greater (p > .05) in G-12SOV (5.8 ± 1.9 and 3.7 ± 1.7) than G-9SOV (3.5 ± 1.1 and 0.8 ± 0.3 respectively). The total follicle count (all follicles ≥2 mm in diameter) at the sixth pFSH dose (at P4 -device removal) was positively correlated (p < .05) with the number of CL (r = .95) and viable embryos (r = .91) in G-12SOV . The ewes with ≥10 Cl (48% of all flushed donors) yielded 80.5% of viable embryos. In summary: (a) Morada Nova ewes from G-12SOV group had better superovulatory responses compared with G-9SOV group; (b) total follicle count at the last pFSH dose was a good predictor of superovulatory responses only in the ewes primed with P4 for 12 days; and (c) animals with ≥10 ovulations are main contributors to viable embryo production in Morada Nova ewes.

Human chorionic gonadotropin affects original (ovulatory) and induced (accessory) corpora lutea, progesterone concentrations, and pregnancy rates in anestrous dairy goats.

Two experiments were conducted in acyclic Alpine (A) and Saanen (S) goats that received intravaginal sponges containing 60 mg of medroxyprogesterone acetate for 6 days, as well as 200 IU of eCG and 30 µg d-cloprostenol i.m. 24 h before sponge removal. On day 7 (day 0 = onset of synchronized estrus), all goats were randomly divided into two groups: animals treated with 300 IU of hCG i.m. (hCG; Exp.1: n = 8A; Exp.2: n = 75A + S) and untreated controls (Control; Exp.1: n = 8A; Exp. 2: n = 70A + S). In Exp.2, all goats were artificially inseminated. Transrectal ovarian ultrasonography and blood collection were done on days 7, 10, 13, 17, and 21 (Exp.1), and pregnancy detection on day 60 (Exp.2). Estrus and ovulations occurred in five hCG and seven Control animals. Accessory CL (aCL) were detected in all hCG does. The total luteal area of ovulatory corpora lutea (oCL) increased (P < 0.05) on day 10 in hCG does and remained greater (P < 0.05) than in Control until day 21. Total and high-velocity color Doppler area were greater (P < 0.05) for oCL of hCG does on days 13 and 17. Progesterone concentrations were greater (P < 0.05) in hCG does from days 13 to 21 and related directly to the total luteal and oCL area for the duration of the study in all does. The pregnancy rate was higher (P < 0.05) in hCG than in Control by 22.5 %. Human chorionic gonadotropin given on day 7 of the synchronized estrous cycle positively affected CL function and pregnancy rates in seasonally anovular dairy goats.

Effects of Using Corn Dried Distillers' Grains with Solubles (cDDGS) as a Partial Replacement for Soybean Meal on the Outcomes of Pig Fattening, Pork Slaughter Value and Quality.

The present study set out to determine the effects of incorporating cDDGS into starter, grower, and finisher diets (containing 5%, 10%, and 15% of cDDGS, respectively) on growth performance, carcass and meat quality, and cost effectiveness of pig fattening. Sixty-four pigs (mean body weight of 15.0 ± 2.1 kg) were divided into two groups (n = 32) and fed a control diet (cereal-soybean meal-based) or cDGGS-containing diets (with soybean meal partially replaced with cDDGS). Live weights of pigs as well as weight gains/daily weight gains across all fattening phases did not differ between the two groups of fattener pigs studied (p > 0.05). Addition of cDDGS decreased feed intake per pig during the grower (p < 0.05) and finisher (p < 0.01) phases, and, as a result, throughout the entire fattening period (254 vs. 245 kg for control and cDDGS groups, respectively; p < 0.01). The feed conversion ratio (FCR) for the entire fattening period was significantly less for cDDGS-fed fatteners (2.77) than for controls (2.91; p < 0.05). Carcass weights, fat thickness, and meatiness did not vary between the two groups of animals (p > 0.05). Loin depth was greater in the cDDGS group by ~5 mm (p < 0.05). Slaughter value was higher for the cDDGS group (76.1% vs. 77.0%, p < 0.05). The total cost of fattening and total cost of 1 kg of body weight decreased in cDDGS compared with the control subset of fatteners by ~7% and 8% during the grower and finisher phases, respectively (p < 0.01). The simplified direct surplus per pig was approximately 63% higher for the cDDGS group. Our results indicate that even moderate inclusion of cDDGS to concentrate mixtures (or a partial replacement of soybean meal with cDDGS) may improve FCR without any substantial changes in meat and back fat characteristics as well as significantly decrease the cost of feeding and increase the profitability of pig production.

NonSurgical Embryo Recovery from Estrus-Synchronized or Superovulated Morada Nova Ewes: A Feasible Strategy for Sheep Embryo Banking.

This study assessed the feasibility of in vivo embryo production and nonsurgical embryo recovery (NSER) in Morada Nova ewes (an endangered native Brazilian breed of sheep) subjected to different estrus synchronization and/or superovulation protocols. Ewes received intravaginal sponges soaked with 60 mg medroxyprogesterone acetate (MAP), which were kept in place for six (G6; n = 12), nine (G9; n = 12), or 12 (G12; n = 12) days. Half of the ewes in each group remained estrus synchronized only (SYNCH) and the other half was superovulated (SOV) with 133 mg porcine follicle-stimulating hormone (pFSH). There were no differences (p > 0.05) in antral follicle counts determined with ultrasonography 60 hours before MAP sponge removal (or at the time of the first pFSH dose) among G6 (6.4 ± 0.9), G9 (6.2 ± 0.7), and G12 (5.5 ± 0.6). Estrus responses and NSER success rates did not vary (p > 0.05) among the three progestin-treatment groups of ewes for either estrus-induced or superovulated animals. The onset of estrus occurred 10-12 hours later (p < 0.01) in G9SYNCH ewes compared with G6SYNCH and G12SYNCH, and the duration of estrus was ∼19 hours greater (p < 0.01) in G9SOV than in G6SOV. The average duration of the NSER procedure was 32.6 ± 1.3 minutes. At least one structure was recovered in 85.7% of synchronized and in 87.5% of superovulated ewes. Viable embryo recovery rates were also similar (p > 0.05) for G6 (1.0 ± 0.3 and 2.5 ± 1.5), G9 (1.3 ± 0.5 and 4.8 ± 2.0), and G12 groups (1.0 ± 0.3 and 4.8 ± 2.3; estrus-synchronized and superovulated ewes, respectively). In conclusion, progestogen pretreatment of different durations and NSER can be employed in Morada Nova ewes, resulting in reasonable viable embryo recovery rates in both estrus-synchronized and superovulated animals. Therefore, both techniques are suitable for use in commercial settings as well as small ruminant conservation programs.

Bovine in vitro embryo production using media prepared with Milli-Q® Water or nanowater.

The aim of this study was to compare the efficiency of in vitro embryo production (IVP) following the collection of bovine ovaries and 22-h in vitro maturation (IVM) of oocytes in media prepared with Milli-Q® Water (n = 509 oocytes) or nanowater (NW; n = 304 oocytes). The mean cleavage (63.8 ± 4.6 % vs. 63.6 ± 6.1 %, respectively; mean ± SEM) and blastocyst formation rate (16.3 ± 3.4 % vs. 16.7 ± 6.7 % of presumptive zygotes, respectively) did not vary (P > 0.05; Student t-test) between the two types of media diluents. NW is a safe substitute for Milli-Q® Water for IVM of bovine oocytes.

Are the spectral Doppler indices of ovarian arteries indicative of antral follicular development and predictive of ovulatory responses and embryo yields in superovulated ewes?

Nineteen ewes received 200 mg of pFSH administered in eight decreasing doses from Days 1 to 4, starting three days before CIDR® device removal. Ten ewes received an injection of 350 µg of estradiol benzoate at CIDR® device insertion (Group E) and nine animals served as controls (Group C). B-mode and spectral Doppler ultrasonographic examinations were performed daily throughout superovulatory treatment to enumerate ovarian antral follicles and to determine ovarian blood flow indices, respectively. There were no differences (P > 0.05) in superovulatory responses between left and right ovaries/uterine horns or the two groups of animals. End-diastolic velocity (EDV) and mean velocity (Vm) values were greater (P < 0.05) on Days 1 and 2, and peak systolic velocity (SVp) was greater (P < 0.05) on Day 3 in Group C than in Group E. In Group E 15 correlations was recorded among indices (SVp, Vm, EDV, flow velocity integral-FVI, and pulsatility index-PI) and follicles numbers in different size classes on Days 1, 2 and 4, and seven correlations among indices (SVp, EDV, Vm, and vascular resistance index-RI) and superovulatory/embryo results (numbers of regressing corpora lutea, numbers/percentages of degenerated embryos and viability rates) on Days 1, 2 and 3. In Group C, there were three correlations among EDV and RI and medium-sized/large follicle numbers on Days 1 and 3, and five correlations among indices (EDV, RI and PI) and superovulatory/embryo results (numbers of luteinized unovulated follicles, degenerated embryos and unfertilized eggs) on Days 2 or 4. There was a lack of consistency in the velocimetric correlates of antral follicle numbers and superovulatory responses between the left and right side. Therefore, the usefulness of ovarian arterial indices to predict ovine superovulatory outcomes remains equivocal and requires further confirmatory studies.

Bovine oviductal fluid (bOF) collected in the follicular or luteal phase of the estrous cycle exerts similar effects on ram sperm kinematics and acrosome reactivity in vitro.

This study examined the effects of bovine oviductal fluid (bOF) obtained during the follicular or luteal phase of the estrous cycle on ram sperm kinematics, capacitation status and plasma membrane (PM) integrity at various time points during the 24-h incubation period. Fresh ram spermatozoa were selected using the swim-up technique and then incubated separately with either follicular phase (FbOF) or luteal phase (LbOF) bovine oviductal fluid added to Fert-TALP medium (positive control - POSControl) or in Fert-TALP medium without capacitating agents (negative control - NEGControl) at 38 °C under 5% CO2. Incubation with FbOF or LbOF for 2 h and 4 h promoted an increase (P <  0.05) in most of the sperm motility parameters as compared with the NEGControl group, and bOF-induced changes in sperm kinematics were similar (P >  0.05) to those seen in the POSControl group. After 6 h of incubation, the stimulatory effect of FbOF or LbOF on ram sperm kinematics was no longer observed (P >  0.05). Sperm PM integrity was not affected (P >  0.05) by incubation in bOF-supplemented media or in absence of capacitating factors (NEGControl). Although neither FbOF nor LbOF had any effect on sperm capacitation rates, the proportion of acrosome-reacted spermatozoa was greater (P < 0.05) for bOF-containing media compared with the NEGControl group during the long incubation periods (18 h and 24 h). In conclusion, bOF from either follicular or luteal phase of the estrous cycle enhances ram sperm motility for up to 4 h and the rate of acrosome reaction after long (18-24 h) incubation periods without affecting sperm viability.

Comparison of the intravenous and intravaginal route of oxytocin administration for cervical dilation protocol and non-surgical embryo recovery in oestrous-induced Santa Inês ewes.

This study compared the effects of intravaginal and intravenous routes of oxytocin (OT) administration in 46 oestrous-induced Santa Inês ewes (6-day treatment with progestin-releasing intravaginal sponges and a single injection of 200 IU of eCG at the time of sponge removal) that underwent transcervical embryo recovery 6-7 days after oestrous onset and mating. All ewes received 37.5 µg of d-cloprostenol via latero-vulvar route, and 1 mg of oestradiol benzoate i.m. 16 hr before and 50 IU of OT 20 min before non-surgical embryo recovery (NSER), with OT being administered intravenously (n = 21) or intravaginally (n = 21). An overall oestrous response was 95.6% (44/46), and adequate cervical retraction could be accomplished in 78.6% (33/42) of ewes. The percentage of successful NSER procedures was 57% (24/42) or 72.7% (24/33) of animals with sufficient cervical retraction. The duration of NSER procedure averaged 28 min (range: 17-40 min) and ~96% of flushing fluid could be recovered (range: 85%-100%). Out of 18 ewes that could not undergo NSER, 12 (66.6%) presented various anatomical barriers, whilst the other 33.4% did not present these barriers and still could not be traversed. Excluding the ewes with those anatomical features, the overall success rate of NSER was 80% (24/30). The route of OT administration had no effect on NSER efficiency or the ease with which transcervical embryo flushing was performed. Both routes of OT administration can be used for cervical dilation protocol. Discarding ewes with anatomical features precluding cervical penetration is highly recommended to increase the efficacy of NSER in sheep.

Echotextural characteristics of the mammary gland during early lactation in two breeds of sheep varying in milk yields.

The main goal of this preliminary study was to determine and compare ultrasonographic characteristics of the mammary gland in two genotypes of ewes varying in milk productivity at 2, 3 and 4 weeks after lambing. Ultrasonographic images of the udder were obtained using the 5.0- and 7.5-MHz transducers, in axial and coronal planes, in four low milk-yielding Polish Mountain sheep and six high milk-yielding Olkuska ewes. All ultrasonograms were subjected to computerized image analyses using commercially available image analytical software (Image ProPlus ® ; Media Cybernetics Inc., San Diego, CA, USA) to determine numerical pixel values (NPVs) and heterogeneity (pixel standard deviation-PSD) of the mammary gland parenchyma. During the 28-day period post-partum, the Olkuska sheep exceeded (P < 0.05) Polish Mountain ewes in milk productivity (31.6 ± 2.7 l and 25.0 ± 4.2 l, respectively; means ± SEMs) as estimated by the mean weight gains of suckling lambs. In animals examined with the 5.0-MHz transducer, mean NPVs of the mammary gland parenchyma in Olkuska ewes and mean PSD in both genotypes of ewes were lower (P < 0.05) before than after milking. In addition, PSD recorded both before and after milking were lower (P < 0.05) in the Polish Mountain compared with Olkuska breed. Mean PSD values for the mammary gland were less (P < 0.05) before than after milking in Polish Mountain ewes and they were greater (P < 0.05) in Olkuska compared with Polish Mountain ewes examined with the 7.5-MHz probe after milking. It can be concluded that milk quantity, histomorphology of the udder and ultrasound transducer frequency may all impinge on the echotextural characteristics of the mammary parenchyma in different breeds of sheep. Our observations warrant future studies of correlations between milk composition, mammary gland histophysiology and ultrasonographic image attributes of the mammary gland in ruminants.

Storage of boar semen at 16-18 °C in the long-term commercial extender prepared with deionized water or nanowater.

The aim of this study was to assess the usefulness of nanowater (NW; water declusterized using cold plasma treatment) as a diluent for a commercial boar semen extender during the 15-day storage (Days 1 to 15) at 16-18 °C. Ejaculates collected from 8 boars were subjected to the standard evaluation and then diluted in the extender prepared with deionized water (DW) or NW to a final concentration of 3×109 spermatozoa/ml. The proportion of defective spermatozoa increased (P<0.05) from Day 10 to Day 15 of storage (22.8±16.6% to 41.8±26.4% in DW group and 18.6±11.7% to 34.8±25.4% in NW group) and it was significantly greater in DW group compared with NW group on Days 5 and 10 due mainly to a greater (P<0.05) number of mid-piece defects in semen stored in the DW-containing extender. Sperm progressive motility decreased (P<0.05) in both groups between Days 2 and 6, Days 6 and 10, and Days 10 and 12, whereas the percentage of motile spermatozoa declined (P<0.05) to Day 14 only in NW group. Sperm motility was greater (P<0.05) in NW group compared with DW group from Day 5 to Day 13. A decline in sperm progressive motility below 40% in all semen samples occurred by Day 11 in DW group and by Day 12 in NW group. The mean survival time of sperm at 37 °C ex situ was greater in NW group than in DW group on Day 5 (314±87 min compared with 284±87 min) and Day 10 (223±34 min compared with 182±27 min; NW group compared with DW group, respectively). There were no differences (P>0.05) between the two groups in the concentrations of alkaline phosphatase and aspartate aminotransferase in semen extender. To summarize, the use of NW as an extender diluent exerts cytoprotective effects on boar spermatozoa and delays a decline in sperm progressive motility.

Combined treatment with oestradiol benzoate, d-cloprostenol and oxytocin permits cervical dilation and nonsurgical embryo recovery in ewes.

This study examined the feasibility of transcervical embryo recovery after the hormonal treatment to induce cervical dilation, following the 7-day oestrous synchronization protocol in multiparous Santa Inês ewes. A total of 23 cyclic ewes received two doses of 37.5 µg of d-cloprostenol by latero-vulvar route 7 days apart. After the second injection of d-cloprostenol, the ewes were checked for oestrus (every 12 hr) and then mated by fertile rams throughout the oestrous period. All ewes received 37.5 µg of d-cloprostenol (latero-vulvar) and 1 mg of oestradiol benzoate by either intramuscular (EBim group; n = 12) or intravaginal (EBivg group; n = 11) route 16 hr before embryo flushing. Twenty minutes before the flushing, 50 IU of oxytocin were administered intravenously. The oestrous response (i.e., the percentage of ewes that showed signs of oestrous behaviour after the second d-cloprostenol injection) was 91.3% (21/23). The proportion of successfully penetrated ewes (81.8% compared with 80.0%), the mean duration of embryo flushing (24.7 ± 2.0 min compared 26.2 ± 1.9 min), the flushing fluid recovery rate (94.8 ± 1.3% compared with 91.0 ± 2.9%) and the average number of structures recovered per ewe (0.5 ± 0.4 compared with 0.8 ± 0.4) did not vary (p > 0.05) between the EBim and EBivg groups. Viable embryos were recovered from 41.2% (7/17) of successfully penetrated ewes. It can be concluded that nonsurgical (i.e., transcervical) embryo collection can be performed in oestrous-synchronized Santa Inês ewes pretreated with d-cloprostenol, oxytocin and oestradiol benzoate, with the latter hormone administered by either the intramuscular or intravaginal route.

Elastographic and echotextural characteristics of foetal lungs and liver during the final 5 days of intrauterine development in dogs.

Objective was to evaluate the echotexture and characteristics during terminal development of canine foetal respiratory and hepatic systems through elastographic examinations. Fifteen pregnant bitches were evaluated by ultrasonography twice daily, from the 53rd gestational day until whelping, and images obtained from 120 to 0 h before parturition were analysed. Images of foetal lungs and liver were recorded and then used for computer-assisted analyses to determine quantitative attributes. Acoustic Radiation Force Impulse (ARFI) elastographic of internal organs were classified as 'soft' (white areas) or 'hard' (dark areas) and quantitative analyses determined the mean shear wave velocities (SWV) of foetal lungs and liver. After delivery, canine neonates were clinically evaluated, and their health status was monitored weekly until 60 days post-partum. Sonographic parameters over time were compared by ANOVA and Pearson's correlations were used to determine associations between SWVs and echotextural variables. Foetal lungs and liver had a homogeneous echotexture and pulmonary parenchyma appeared hyperechoic when compared with that of the liver. Mean numerical pixel values (NPVs) of lungs decreased from 120 to 24 h and subsequently increased until parturition (P = 0.04). Lungs and liver mean (± SD) SWVs (0.98 ± 0.12 and 0.84 ± 0.11 m/s, respectively) didn't vary (P > 0.05) over time. Fluctuations in pulmonary NPVs indicated there was a pattern corresponding to structural and functional changes that occur during the terminal stage of pre-natal canine development and hence can be a useful diagnostic tool in veterinary. Foetal lung and liver SWVs were relatively consistent and there was no detectable changes during the pre-partum period for this variable or in echotexture.