Effect of a puberty induction protocol based on injectable long acting progesterone on pregnancy success of beef heifers serviced by TAI.

Induction protocols based on progesterone (P4) are used prior to a synchronization program for timed-AI (TAI) to increase number of pubertal heifers and pregnancy per AI (P/AI). Injectable, long-lasting P4 (iP4) is a novel, practical method to supplement P4 in cattle. Here, we aimed to test the effect of an induction protocol based on a single injection of iP4 on P/AI of heifers. Bos indicus (Nellore) heifers were classified as pubertal (PUB; n = 224) or prepubertal (PRE; n = 414) based on two ovarian ultrasonographyc exams conducted 10 d apart. Heifers with a corpus luteum (CL) in any of the exams were considered PUB. Within each puberty status, heifers were assigned to receive nothing (NoiP4) or an induction protocol (iP4). Induction consisted of a single injection of 150 mg of iP4 on D-31, followed by injections of 1 mg of estradiol benzoate (EB) and 150 µg of prostaglandin analogue (PGF) on D-21. On D-9, all heifers received 2 mg of EB + 75 µg of PGF associated to intravaginal P4-device insertion. On D-3, P4-releasing devices were removed and 150 µg of PGF injected. Heifers were inseminated based on estrus on D-1 or were TAI on D0. On D0, all heifers received a dose of GnRH analogue. On D-21, iP4 treatment stimulated a 50% increase in the uterine score (UTS) and a 19% increase in the diameter of the largest follicle of PRE heifers (P < 0.01). On D-9, PRE|iP4 group had a greater proportion (P < 0.01) of CL (63.3%) than PRE|NoiP4 group (11.6%). On D-3, exposure to 6 d P4-releasing device stimulated UTS of PRE|NoiP4 group in a similar fashion than the induction protocol, but it did not have any additional positive effect for PRE|iP4 heifers. P/AI of PRE|iP4 group was similar to that of the PUB groups (44.7 vs 46.9%), but was more than that of PRE|NoiP4 (34.2%). There was an overall 7.7% increment (P = 0.07) on P/AI of iP4 treated heifers (iP4: 46.0% vs. NoiP4: 38.3%). In conclusion, implementation of an induction protocol based on iP4 was efficacious to hasten puberty. Induction stimulated uterine development and follicular growth of prepubertal heifers, ultimately leading to pregnancy success similar to that of pubertal heifers.

Increased pregnancy rate in beef heifers resynchronized with estradiol at 14 days after TAI.

We aimed to evaluate the treatment with estradiol benzoate (EB) or 17ß-estradiol (E2) associated with progesterone (P4) for resynchronization of ovulation 14 days after timed artificial insemination (TAI). In Experiment 1 (Exp. 1), Nelore heifers were submitted to TAI (D0). On D14, the animals received an intravaginal P4 device and were randomly assigned to one of three groups: control (no treatment; n = 17); EB (1  mg EB; n = 17); and E2+P4 (1 mg E2 + 9 mg P4; n = 18). Ultrasonography evaluations were performed daily from D14 to D22 to map follicular and luteal dynamics. On D22, the P4 devices were removed and non-pregnant (NP) animals were determined using corpus luteum blood flow Doppler ultrasonography. In Exp. 2, 1295 beef heifers were resynchronized and randomly allocated to the same experimental groups as described in Exp. 1. On D22, the largest follicle (LF) was measured in NP and a second TAI was performed on D24. In a subset of heifers (n = 337), an estrus detection patch was used between D22 and D24 to monitor estrus expression and the LF was measured at D24. Confirmatory diagnosis of pregnancy was performed between D37-67 and D43-67 after first and second TAI, respectively. In Exp 1, the proportion of heifers with a synchronized follicular wave emergence (from 3 to 5 days after treatment) was greater (P < 0.05) in the EB group (93.8%) than in the control (62.5%) and E2+P4 (64.7%) groups. Structural luteolysis occurred earlier (P < 0.05) in the EB and E2+P4 groups than in the controls. The pregnancy rate after first TAI did not differ (P > 0.1) among the groups at D22 and at confirmatory diagnosis in both experiments. In Exp 2, the potential pregnancy loss between D22 and D37-67 was similar (P > 0.1) in the control (19% [36/185]), EB (15% [28/182]) and E2+P4 (15% [28/184]) groups. The LF diameter (mm) on D22 was greater (P < 0.05) in the control group (11.9 ± 0.1) than in EB (11.3 ± 0.1) and E2+P4 (11.5 ± 0.1). No difference (P > 0.1) was observed in the proportion of heifers detected in estrus, but LF growth rate (mm/day) between D22 and D24 was greater (P < 0.05) in EB group (0.9 ± 0.08) than in control (0.6 ± 0.07) and E2+P4 (0.7 ± 0.09) groups. The pregnancy rate for the second TAI was greater (P < 0.05) in the EB group (47% [94/200]) than in the control (37% [76/203]), but did not differ (P > 0.1) from the E2+P4 group (43% [93/214]). In conclusion, the treatment with 1 mg EB or 1 mg E2 + 9 mg P4 at 14 days post-TAI anticipates luteolysis in NP heifers but does not compromise pregnancy. The EB treatment induces a new synchronized follicle wave emergence and increases the pregnancy rate of resynchronized NP heifers.

Aspects related to the technique and the utilization of sexed semen in vivo and in vitro

The development of methods capable of selecting the sex of animals has always been a great challenge for humankind. Separating X chromosome- bearing and Y chromosome-bearing sperm based on DNA difference using flow cytometry is the only technique that has achieved a useful level of progress, especially in cattle. This allowed the technique to be commercially applicable in this species. Thorough this review, aspects related to the sexing technique (flow cytometry) and to the sperm will be discussed along with the in vitro use of sexed semen. Besides, number of sperm used and semen deposition location, the influence of reproductive status (heifers vs. cows), time of insemination and production of embryos in superovulated donors will be reviewed.

Aspects related to the technique and the utilization of sexed semen in vivo and in vitro

The development of methods capable of selecting the sex of animals has always been a great challenge for humankind. Separating X chromosome- bearing and Y chromosome-bearing sperm based on DNA difference using flow cytometry is the only technique that has achieved a useful level of progress, especially in cattle. This allowed the technique to be commercially applicable in this species. Thorough this review, aspects related to the sexing technique (flow cytometry) and to the sperm will be discussed along with the in vitro use of sexed semen. Besides, number of sperm used and semen deposition location, the influence of reproductive status (heifers vs. cows), time of insemination and production of embryos in superovulated donors will be reviewed.(AU)

Métodos de avaliação da morfologia e função espermática: momento atual e desafios futuros / Methods of the assessment of morphology and function of sperm: actual moment and future challenges

Com o propósito de obter resultados que demonstrem maior repetibilidade na avaliação da morfologia tanto quanto na função espermática, diversas técnicas laboratoriais tem sido desenvolvidas. Surgiram sistemas que utilizam a análise computadorizada de imagens, métodos de “coloração” empregando corantes fluorescentes (sondas fluorescentes) em microscopia de epifluorescência ou citometria de fluxo, que aumentaram a possibilidade de uma análise mais criteriosa da integridade estrutural dos espermatozóides. Os autores descrevem experiências, por mais de uma década, com a utilização destas biotécnicas, fazendo uma análise crítica, bem como apontando os desafios futuros, inclusive na área de biologia molecular espermática.(AU)

In order to obtain results showing greater repeatability in morphology and sperm function assessment, several laboratorial techniques have been developed continuously. Systems that utilize computerized image analysis, staining methods using fluorescent dies (fluorescent probes) in epifluorescence microscopy or flow cytometry, that allowed a more judicious evaluation of sperm structure integrity arose. Authors describe experiences, over a decade, with the use of these biotechiques, critically analyzing and pointing out future challenges, including the field of sperm molecular biology.(AU)

Métodos de avaliação da morfologia e função espermática: momento atual e desafios futuros / Methods of the assessment of morphology and function of sperm: actual moment and future challenges

Com o propósito de obter resultados que demonstrem maior repetibilidade na avaliação da morfologia tanto quanto na função espermática, diversas técnicas laboratoriais tem sido desenvolvidas. Surgiram sistemas que utilizam a análise computadorizada de imagens, métodos de “coloração” empregando corantes fluorescentes (sondas fluorescentes) em microscopia de epifluorescência ou citometria de fluxo, que aumentaram a possibilidade de uma análise mais criteriosa da integridade estrutural dos espermatozóides. Os autores descrevem experiências, por mais de uma década, com a utilização destas biotécnicas, fazendo uma análise crítica, bem como apontando os desafios futuros, inclusive na área de biologia molecular espermática.

In order to obtain results showing greater repeatability in morphology and sperm function assessment, several laboratorial techniques have been developed continuously. Systems that utilize computerized image analysis, staining methods using fluorescent dies (fluorescent probes) in epifluorescence microscopy or flow cytometry, that allowed a more judicious evaluation of sperm structure integrity arose. Authors describe experiences, over a decade, with the use of these biotechiques, critically analyzing and pointing out future challenges, including the field of sperm molecular biology.