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Maternal nutrient restriction (NR) causes small for gestational age (SGA) offspring, which are at higher risk for accelerated postnatal growth and developing insulin resistance in adulthood. Skeletal muscle is essential for whole-body glucose metabolism, as 80% of insulin-mediated glucose uptake occurs in this tissue. Maternal NR can alter fetal skeletal muscle mass, expression of glucose transporters, insulin signaling, and myofiber type composition. It also leads to accumulation of intramuscular triglycerides (IMTG), which correlates to insulin resistance. Using a 50% NR treatment from gestational day (GD) 35 to GD 135 in sheep, we routinely observe a spectral phenotype of fetal weights within the NR group. Thus, we classified those fetuses into NR(Non-SGA; n = 11) and NR(SGA; n = 11). The control group (n = 12) received 100% of nutrient requirements throughout pregnancy. At GD 135, fetal plasma and gastrocnemius and soleus muscles were collected. In fetal plasma, total insulin was lower in NR(SGA) fetuses compared NR(Non-SGA) and control fetuses (P < 0.01), whereas total IGF-1 was lower in NR(SGA) fetuses compared with control fetuses (P < 0.05). Within gastrocnemius, protein expression of insulin receptor (INSRB; P < 0.05) and the glucose transporters, solute carrier family 2 member 1 and solute carrier family 2 member 4, was higher (P < 0.05) in NR(SGA) fetuses compared with NR(Non-SGA) fetuses; IGF-1 receptor protein was increased (P < 0.01) in NR(SGA) fetuses compared with control fetuses, and a lower (P < 0.01) proportion of type I myofibers (insulin sensitive and oxidative) was observed in SGA fetuses. For gastrocnemius muscle, the expression of lipoprotein lipase (LPL) messenger RNA (mRNA) was upregulated (P < 0.05) in both NR(SGA) and NR(Non-SGA) fetuses compared with control fetuses, whereas carnitine palmitoyltransferase 1B (CPT1B) mRNA was higher (P < 0.05) in NR(Non-SGA) fetuses compared with control fetuses, but there were no differences (P > 0.05) for protein levels of LPL or CPT1B. Within soleus, there were no differences (P > 0.05) for any characteristic except for the proportion of type I myofibers, which was lower (P < 0.05) in NR(SGA) fetuses compared with control fetuses. Accumulation of IMTG did not differ (P > 0.05) in gastrocnemius or soleus muscles. Collectively, the results indicate molecular differences between SGA and Non-SGA fetuses for most characteristics, suggesting that maternal NR induces a spectral phenotype for the metabolic programming of those fetuses., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Maternal nutrient restriction causes small for gestational age (SGA) offspring, which exhibit a higher risk for metabolic syndrome in adulthood. Fetal skeletal muscle is particularly sensitive to maternal nutrient restriction, which impairs muscle mass and metabolism. Using a 50% nutrient restriction treatment from gestational day (GD) 35 to GD 135 in sheep, we routinely observe a spectral phenotype of fetal weights within the nutrient-restricted (NR) group. Thus, our objective was to evaluate the effect of maternal NR on muscle mass, myofiber hypertrophy, myonuclear dotation, and molecular markers for protein synthesis and degradation, while accounting for the observed fetal weight variation. Within the NR group, we classified upper-quartile fetuses into NR(Non-SGA) (n = 11) and lower-quartile fetuses into NR(SGA) (n = 11). A control group (n = 12) received 100% of nutrient requirements throughout pregnancy. At GD 135, fetal plasma and organs were collected, and gastrocnemius and soleus muscles were sampled for investigation. Results showed decreased (P < 0.05) absolute tissue/organ weights, including soleus and gastrocnemius muscles, in NR(SGA) fetuses compared to NR(Non-SGA) and control. Myofiber cross-sectional area was smaller in NR(SGA) vs control for gastrocnemius (P = 0.0092) and soleus (P = 0.0097) muscles. Within the gastrocnemius muscle, the number of myonuclei per myofiber was reduced (P = 0.0442) in NR(SGA) compared to control. Cortisol may induce protein degradation. However, there were no differences in fetal cortisol among groups. Nevertheless, for gastrocnemius muscle, cortisol receptor (NR3C1; P = 0.0124), and FOXO1 (P = 0.0131) were upregulated in NR(SGA) compared to control while NR(Non-SGA) did not differ from the other 2 groups. KLF15 was upregulated (P = 0.0002) in both NR(SGA) and NR(Non-SGA); while FBXO32, TRIM63, BCAT2 or MSTN did not differ. For soleus muscle, KLF15 mRNA was upregulated (P = 0.0145) in NR(SGA) compared to control, and expression of MSTN was increased (P = 0.0259) in NR(SGA) and NR(Non-SGA) compared to control. At the protein level, none of the mentioned molecules nor total ubiquitin-labeled proteins differed among groups (P > 0.05). Indicators of protein synthesis (total and phosphorylated MTOR, EI4EBP1, and RPS6KB1) did not differ among groups in either muscle (P > 0.05). Collectively, results highlight that maternal NR unequally affects muscle mass in NR(SGA) and NR(Non-SGA) fetuses, and alterations in myofiber cross-sectional area and myonuclei number partially explain those differences., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies-an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4-5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.

The objective was to compare a proprietary egg yolk-based cryopreservation medium with a chemically defined soy-based medium, as well as to examine effects of temperature of glycerol addition on sperm parameters and IVF after freezing and thawing of domestic cat sperm. Semen was collected from adult cats (four males and three ejaculates per male), divided in four equal aliquots, and extended in either egg yolk with 4% glycerol added before (EYG) or after (EY) cooling to 5 °C, or soy-lecithin with 4% glycerol added before (SLG) or after (SL) cooling to 5 °C. Extended sperm were frozen in straws over liquid nitrogen vapor. Sperm progressive motility (%) and rate of progressive movement (scale of 0-5) were evaluated at 0, 1, 3, 6, and 24 h post-thaw. Sperm capacitation, acrosome integrity, and DNA integrity were assessed at 15 min post-thaw. Effects of media (EY or SL) on IVF success was also examined (three males and three ejaculates per male). Sperm motility was greater (P < 0.05) in soy-based compared with egg yolk-based media at 3, 6, and 24 h post-thaw. A higher (P < 0.05) percentage of noncapacitated sperm (pattern F) were present in soy-based (SLG, 63.7 ± 9.2%; and SL, 64.1 ± 9.2%) compared with egg yolk-based (EYG, 49.9 ± 7.9%; and EY, 52.4 ± 18.6%) cryopreservation media, regardless of temperature of glycerol addition. Addition of glycerol at 5 °C increased (P < 0.05) percentage of sperm motility at 6 h (EYG 16.3 ± 8.3% vs. EY, 24.0 ± 11.7%; SLG, 36.7 ± 6.5% vs. SL, 42.9 ± 10.1%) and 24 h (EYG, 2.1 ± 3.3% vs. EY, 8.3 ± 3.9%; SLG, 11.3 ± 8.3% vs. SL, 18.8 ± 7.4%) post-thaw in both media. There were no differences (P > 0.05) between cryodiluents in embryo cleavage, percentage of embryos reaching blastocyst, or cell number per blastocyst. The chemically defined, soy-based medium resulted in better preservation of long-term motility and capacitation status of frozen-thawed domestic cat sperm compared with a commercial egg yolk-based extender, without compromising fertilizing ability., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Artificial insemination (AI) is potentially invaluable as an adjunct to natural breeding for the conservation management of non-domestic felid populations. The efficacy of AI, however, must be substantially improved for applied use, especially when using frozen semen. Our recent advances in using laparoscopic oviductal AI (LO-AI) with low sperm numbers and freezing of cat semen in a soy lecithin-based cryoprotectant medium suggest that combining these two approaches might improve pregnancy outcomes with frozen-thawed spermatozoa. In this study, our objectives were to (i) assess the effect of two gonadotropin dosages (100 vs 150 IU eCG) on ovarian response in domestic cats and (ii) compare the relative fertility of frozen-thawed and fresh semen in vivo following LO-AI. All 16 females ovulated after gonadotropin treatment and were inseminated with fresh semen from one male and frozen-thawed semen from a second male. There were no differences between gonadotropin dosages in CL number, pregnancy percentage or litter size. Half (8/16) of the females conceived, with seven females giving birth to a total of 36 offspring. Paternity analysis showed that more kittens resulted from LO-AI with fresh (28/36, 78%) than frozen-thawed (8/36, 22%) semen, possibly due to impaired motility and longevity of thawed sperm. These results demonstrated that viable offspring can be produced by AI using semen frozen in a soy lecithin-based medium. Insemination with greater numbers of frozen-thawed spermatozoa, combined with further refinement of cat sperm cryopreservation methods, may be necessary to optimize pregnancy success with LO-AI in domestic and nondomestic cats., (© 2012 Blackwell Verlag GmbH.)