Soybean lecithin-based extender as an alternative for goat sperm

cryopreservation
Original Research Article
Pages 47-51
Andra Helena Vidal, Andr Mariano Batista, Ellen Cordeiro Bento da Silva, Wilton Arruda Gomes, Marina Arruda
Pelinca, Sildivane Valccia Silva, Maria Madalena Pessoa Guerra
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Abstract
The aim of this study was to evaluate the effect of different concentrations of soybean lecithin (SL) in extenders for
sperm goat cryopreservation. Sexually mature male Saanen goats (n = 4) were used, and the ejaculates were
obtained using an artificial vagina method. The semen samples were pooled and diluted in a skim milk-based
extender (control group; CG) or Tris extender supplemented with SL at different concentrations (G1 = 0.04%, SL
G2 = 0.08% SL and G3 = 0.16%) for a final concentration of 240 106 spermatozoa/mL. The semen samples were
packed in straws (0.25 mL), frozen using an automated system and stored in liquid nitrogen (196 C). After thawing
(37 C/30 s), the samples were evaluated for sperm quality parameters, including sperm motility, membrane integrity,
acrosome integrity and mitochondrial activity. No significant difference was observed among the experimental and
control groups for all of the parameters (P > 0.05). However, even though the control group presented a significantly
lower mitochondrial membrane potential compared to fresh semen (P < 0.05), the same did not occur for the
extender supplemented with soybean lecithin, that is, it did not differ from fresh sperm (P > 0.05). The extender
containing soybean lecithin at different concentrations preserved the sperm quality parameters in a manner similar to
the conventional skim milk-based extender. Thus, it is concluded that an extender containing soybean lecithin as the
lipoprotein source can be used for freezing goat semen.

Article Outline

1. Introduction

2. Materials and methods

2.1. Chemicals

2.2. Animals and semen collection

2.3. Analyses and semen freezing

2.4. Thawing and in vitro sperm analysis

2.4.1. Acrosomal integrity

2.4.2. Membrane integrity

2.4.3. Mitochondrial membrane potential

2.5. Statistical analysis

3. Results

4. Discussion

5. Conclusion

References

In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation
of bovine semen Original Research Article
Theriogenology, Volume 60, Issue 2, July 2003, Pages 269-279
Viviana A. Aires, Klaus-Dieter Hinsch, Frank Mueller-Schloesser, Katja Bogner, Susanne Mueller-Schloesser, Elvira
Hinsch
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Abstract
Semen diluents containing egg yolk as a cryoprotectant may pose hygienic risks and are difficult to standardize.
Although a new generation of semen diluents free of animal ingredients is available, egg yolk-containing extenders
are still widely used for cryopreserving semen. We compared the effects of using different extenders on bovine sperm
function in vitro and on fertility in vivo. A soy lecithin extender (SL; AndroMed) and an egg yolk-containing (TRIS-EY)
extender were tested. No differences (P>0.05) were detected between the two extenders for spermzona pellucida
binding capacity (HZI=11513). Assessment of the inducibility of the acrosome reaction with progesterone showed no
differences (P>0.05) between extenders for live acrosome-reacted sperm (152.36 and 14.422.02%, respectively,
for SL and TRIS-EY). However, post-thaw sperm motility was significantly lower (P<0.05) when semen was extended
in the TRIS-EY diluent. Field trials revealed that nonreturn rates of SL-extended semen showed significantly higher
insemination success (P<0.0001) compared with the nonreturn rates for the TRIS-EY extender (70.45 and 67.85%,
respectively). We suggest that consistent with quality standards that should be required for cryoprotectant media and
because of the superior quality of the egg yolk-free extender, a defined soybean lecithin-containing diluter might be
the better choice as a semen extender in the future.

Article Outline

1. Introduction

2. Materials and methods

2.1. Semen collection and preparation for in vitro tests

2.2. Motility analysis

2.3. Capacitation

2.4. Induction of the acrosome reaction with progesterone

2.5. Assessment of viability and acrosomal status

2.6. HZA

2.7. Field study

2.8. Statistical analysis

3. Results

4. Discussion

Effect of antioxidants on post thaw microscopic, oxidative stress parameter and fertility of Boer goat
spermatozoa in Tris egg yolk glycerol extenderOriginal Research Article
Animal Reproduction Science, Volume 136, Issues 12,December 2012, Pages 55-60
Akeel Ahmed Memon, H. Wahid, Y. Rosnina, Y.M. Goh, M. Ebrahimi, F.M. Nadia
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Abstract
This study was conducted to determine the effect of antioxidants on standard semen parameters, lipid peroxidation
and fertility of Boer goat semen after cryopreservation. Ejaculates from four bucks were collected, evaluated and
pooled at 37 C. The pooled semen was diluted with Tris citric acid fructose for washing. Semen samples, which were
diluted with a Tris-based extender containing the antioxidant ascorbic acid (8.5 mg/ml), butylated hydroxytoluene
(2 mM), cysteine (5 mM) and hypotaurine (10 mM) and an extender without antioxidant supplementation were cooled
to 4 C and frozen in 0.25 straws with programmable freezer and finally stored in liquid nitrogen. Data (10 replicates)
were analyzed by one-way analysis of variance. Mean (SEM) progressive motility was significantly higher in
ascorbic acid than other supplement groups and control samples (P > 0.05). Best values were observed in ascorbic
acid followed by BHT, cysteine, and hypotaurine. Antioxidant supplementation in extender showed significant
(P < 0.05) better values than the control group for sperm membrane integrity, acrosome integrity and viability. The
ability of antioxidants to reduce the lipid peroxidation (LPO) after freeze thawing was measured by the formation of
malondialdehyde (MDA) using the thiobarbituric acid method. Results showed that addition of antioxidants
significantly reduced the rate of LPO in comparison to control (P < 0.05). Ascorbic acid exhibited better values
(1.27 0.28), than butylated hydroxytoluene, cysteine and hypotaurine 1.32 0.42, 2.27 0.16 and 2.38 0.17
respectively, which are significantly better than control (3.52 0.54). Higher pregnancy rate was observed with
ascorbic acid followed by butylated hydroxtolune, hypotaurine and cysteine. However, differences in the fertility rate
were non-significant with hypotaurine, cysteine and control groups.

Article Outline

1. Introduction

2. Materials and methods

2.1. Animals

2.2. Semen processing and evaluation

2.3. Processing of semen

2.3.1. Post thaws evaluation

2.4. Malondialdehiyde (MDA) concentrations

2.5. Fertility assessment

2.5.1. Estrus synchronization

2.5.2. Artificial insemination

2.5.3. Pregnancy diagnosis and fertility rate

2.6. Statistical analysis

Recovery and cryopreservation of epididymal sperm from agouti (Dasiprocta aguti) using powdered coconut
water (ACP-109c) and Tris extendersOriginal Research Article
Theriogenology, Volume 76, Issue 6, 1 October 2011,Pages 1084-1089
M.A. Silva, G.C.X. Peixoto, E.A.A. Santos, T.S. Castelo, M.F. Oliveira, A.R. Silva
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Abstract
The objective was to compare the use of powdered coconut water (ACP-109c; ACP Biotecnologia, Fortaleza, CE,
Brazil) and Tris extenders for recovery and cryopreservation of epididymal sperm from agouti. The caudae
epididymus and proximal ductus deferens from 10 sexually mature agoutis were subjected to retrograde washing
using ACP-109c (ACP Biotecnologia) or Tris. Epididymal sperm were evaluated for motility, vigor, sperm viability,
membrane integrity, and morphology. Samples were centrifuged, and extended in the same diluents plus egg yolk
(20%) and glycerol (6%), frozen in liquid nitrogen, and subsequently thawed at 37C for 1 min, followed by reevaluation of sperm characteristics. The two extenders were similarly efficient for epididymal recovery, with regard to
the number and quality of sperm recovered. However, for both extenders, sperm quality decreased (P < 0.05) after
centrifugation and dilution. After sperm cryopreservation and thawing, there were (mean SEM) 26.5 2.6% motile
sperm with 2.6 0.2 vigor in the ACP-109c (ACP Biotecnologia) group, which was significantly better than 9.7 2.6%
motile sperm with 1.2 0.3 vigor in Tris. In conclusion, agouti epididymal sperm were successfully recovered using
either ACP-109c (ACP Biotecnologia) or Tris extenders; however, ACP-109c (ACP Biotecnologia) was a significantly
better extender for processing and cryopreserving these sperm.

Article Outline

1. Introduction

2. Materials and methods

2.1. Animals

2.2. Obtaining epididymal sperm

2.3. Sperm evaluation

2.4. Sperm processing

2.5. Sperm cryopreservation

2.6. Statistical analysis

3. Results

3.1. Recovered and centrifuged sperm characteristics

3.2. Postthaw sperm characteristics

Discussion

Acknowledgments

References

Effect of Vitamin E supplement in diet on antioxidant ability of testis in Boer goat

Original Research

Article
Animal Reproduction Science, Volume 117, Issues 12,January 2010, Pages 90-94
Zhu Hong, Luo Hailing, Meng Hui, Zhang Guijie, Yan Leyan, Yue Dubing
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Abstract
The aim of this study was to evaluate the supplementation of Vitamin E in diet on the antioxidant capacity of testis in
Boer goat. Twenty-four healthy, Boer male kids of similar body weight (BW) were selected at 3 months of age from
the kid flock. Kids were born from does treated with simultaneous flushing and artificial insemination technology. The
Boer kids were divided into four groups randomly, supplemented with 0, 80, 320 and 880 IU kid1 d1 Vitamin E, which
were labeled as Groups 1, 2, 3 and 4, respectively, for 150 days (5 months). Blood samples were collected at the
15th-, 30th-, 60th-, 90th-, 120th-, and 150th-day during the experimental period, and the serums were used to
determine Vitamin E content. Three Boer goats in each group were slaughtered at the age of eight months at the end
of the experiment. Liver and testis were collected to test the Vitamin E content and the antioxidant capacity of testis.
Results showed that the content of Vitamin E in serum, liver and testis increased with the increasing addition of
Vitamin E. However, the content of Vitamin E in the serum, liver and testis, in the control, was significantly lower than
in Groups 2 and 3, respectively, but there was no significant difference between the control Group and Group 4.
When high levels of Vitamin E (880 IU kid1 d1) were added, contents of Vitamin E in serum, liver and testis were
decreased and compared with the controls. Adding a low level (80 IU kid1 d1) of Vitamin E can increase activity of
total anti-oxidation competence (T-AOC) and superoxide dismutase (SOD), and decrease content of nitric oxide (NO)
in testis. MDA (malondialdehyde) content was decreased significantly in Group 3 (P < 0.05). Supplementing a low
level (80 IU kid1 d1) and middle level (320 IU kid1 d1) of Vitamin E decreased activity of nitric oxide syntha (NOS) in
testis (P < 0.05). Vitamin E can increase activity of GSH-PX (glutathione peroxidase). These results indicate that
supplementing Vitamin E protects testis from damage by preoxidation.

Article Outline

1. Introduction

2. Material and methods

2.1. Materials and animals

2.2. Animals management

2.3. Methods

2.4. Statistical analyses

3. Results

3.1. Effect of Vitamin E supplemented in diet on Vitamin E content of serum, liver and testis in

o
Boer goat
o

3.2. Effect of Vitamin E supplemented in diet on antioxidant abilities of testis in Boer goat
4. Discussion

Effect of vitamin E supplementation on development of reproductive organs in Boer goat

Original

Research Article
Animal Reproduction Science, Volume 113, Issues 14,July 2009, Pages 93-101
Zhu Hong, Luo Hailing, Meng Hui, Zhang Guijie
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Abstract | Figures/Tables | References

Abstract
The aim of this study was to evaluate the supplementation of Vitamin E in diet on development of reproductive organs
in Boer goat. Twenty-four healthy, Boer male kids of similar body weight (BW) were selected at 3 months of age from
the kid flock. The Boer kids were randomly divided into four groups and each group was supplemented with Vitamin E
at 0, 80, 320 or 880 IU kid1 d1 for 5 months. Three Boer goats in each group were slaughtered at the age of 8
months and measured for testis and epididymises and a ductus deferens biopsy by H&E stained slides was run. The
results showed that supplementing 80 and 320 IU kid1 d1 Vitamin E increased weights of the epididymis and the
numeric density of convoluted seminiferous tubules. Diameters of convoluted seminiferous tubules and epididymis
ductules and numeric density of spermatogenic cells tended to be larger in 80 IU kid1 d1compared to the control
group (P < 0.05). Weight of ductus deferens and thickness of epididymis wall were increased significantly with
320 IU kid1 d1 (P < 0.01). However, no significant effects were observed on thickness of germinal epithelium,
numeric density of leydig cells, testis weight or indexes of ductus deferens (P > 0.05). In conclusion, supplementing
80 and 320 IU kid1 d1 Vitamin E in diets significantly simulates the development of reproductive organs in Boer
goats. No significantly improved effects were found from supplementing 880 IU kid1 d1.

Article Outline

1. Introduction

2. Material and methods

2.1. Materials

2.2. Animals

2.3. Animals management

2.4. Methods

2.5. Statistical analyses

3. Results

3.1. Effect of Vitamin E supplementation on weight of reproductive organs

3.2. Effect of Vitamin E supplementation on testis indexes

3.3. Effect of Vitamin E supplementation on epididymis indexes

3.4. Effect of Vitamin E supplementation on ductus deferens indexes

4. Discussion

4.1. Effect of Vitamin E supplementation on weight of reproductive organs

4.2. Effect of Vitamin E supplementation on testis indexes

4.3. Effect of Vitamin E supplementation on cell indexes

4.3.1. Leydig cell

4.3.2. Sertoli cells

4.3.3. Spermatogenetic cell

4.4. Effect of Vitamin E supplementation on epididymis wall thickness and diameter of

o
epididymis

4.5. Effect of Vitamin E supplementation on ductus deferens indexes

5. Conclusions

Effect of glutathione in soybean lecithin-based semen extender on goat semen quality after freezethawingOriginal Research Article
Small Ruminant Research, In Press, Corrected Proof,Available online 16 January 2013
Hossein Salmani, Mohammad Mahdi Nabi, Hossein Vaseghi-Dodaran, Mohammad Bozlur Rahman, Abdollah
Mohammadi-Sangcheshmeh, Malek Shakeri, Armin Towhidi, Ahmad Zare Shahneh, Mahdi Zhandi
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Abstract | Figures/Tables | References

Abstract
This study was conducted to investigate the effect of glutathione in a soybean lecithin-based semen extender on
post-thawed goat semen quality. A total of 20 ejaculates were collected from four Mahabadi goats and diluted with
four extenders: (1) egg yolk tris-based extender (EYT), (2) soybean lecithin-based extender (SL0), (3) soybean
lecithin-based extender containing 5 mM glutathione (SL5), and (4) soybean lecithin-based extender containing
10 mM glutathione (SL10) and the experiment was replicated for five times. Sperm motility and motion parameters,
the percent of normal sperm, plasma membrane integrity, viability, and the levels of malondialdehyde (MDA) were
determined after freeze-thawing. Glutathione at the level of 10 mM provided the lowest percent of total and
progressively motile sperm (45 2.94, 25.8 1.81). The percent of plasma membrane intact sperm was lower in SL5
(55.44 2.4) and SL10 (51.52 2.4) than that of EYT (64.27 2.4) and SL0 (62.93 2.4). Sperm viability was
significantly decreased in SL10 (57.98 2.27) in comparison with EYT (66.89 2.27). The percent of normal sperm
was not significantly different between groups. Also, the MDA level was lower in SL5 (1.61 0.29) than that of other
groups. The results of this study showed that most of the post-thawed sperm quality parameters were not significantly
different between EYT and SL0. Moreover, addition of 5 and 10 mM glutathione in a soybean lecithin-based extender
could not improve goat sperm quality after freeze-thawing. In conclusion, it seems that soybean lecithin-based
extender can be a suitable replacement for egg yolk tris-based extender, but addition of glutathione at 5 and 10 mM
cannot fortify the soybean lecithin-based extender for goat semen cryopreservation.

Article Outline

1. Introduction

2. Materials and methods

2.1. Chemicals

2.2. Animals and semen collection

2.3. Semen extending, freezing and thawing

2.4. Semen evaluation

2.4.1. Assessment of sperm motility by CASA

2.4.2. Hypo-osmotic swelling test (HOST)

2.4.3. Assessment of viability

2.4.4. Assessment of sperm morphology

2.4.5. MDA concentration

2.5. Statistical analysis

3. Results

3.1. Sperm motility

3.2. Sperm quality (viability, HOST, normality) parameters and MDA test

4. Discussion

5. Conclusions

Trehalose and glycerol have a dose-dependent synergistic effect on the post-thawing quality of
ram semen cryopreserved in a soybean lecithin-based extender Original Research Article
Cryobiology, In Press, Uncorrected Proof, Available online 13 March 2013
Abozar Najafi, Mahdi Zhandi, Armin Towhidi, Mohsen Sharafi, Abbas Akbari Sharif, Mahdi Khodaei
Motlagh, Felipe Martinez-Pastor
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Abstract | Figures/Tables | References

Abstract
The objective of this study was to examine the interaction of different concentrations of trehalose [0 (T0),
50 (T50) or 100 (T100) mM] and glycerol [5% (G5) or 7% (G7)] on post-thawed quality of ram semen,
cryopreserved in a soybean lecithin (SL)-based extender. Twenty-eight ejaculates were collected from
four rams and diluted with six trehalose/glycerol combinations: T0G5, T50G5, T100G5, T0G7, T50G7,
and T100G7. Sperm motility (CASA), membrane integrity (eosin/nigrosin) and functionality (HOST),
abnormal forms, capacitation status (CTC), mitochondrial activity (rhodamine 123), apoptotic features
(Annexin V/propidium iodide) and lipoperoxidation (malondialdehyde production) were evaluated after
thawing. Extender T100G5 yielded the highest results for total and progressive motility, sperm velocity,
normal morphology, functional membranes, active mitochondria and membrane integrity, with P < 0.05
in general, except for T50G7 (P > 0.05). The combinations T0G5, T0G7 and T100G7 yielded the lowest
post-thaw quality. We could not detect significant changes in other kinematic parameters, capacitation
status or lipoperoxidation. We conclude that, in our SL-based extender, a combination of 100 mM
trehalose and 5% glycerol was the most adequate combination to achieving post-thawing quality in our
soybean lecithin-based extender, and our results support that a synergistic effect among trehalose and
glycerol exists. We suggest that other combinations could improve these results.

Article Outline
Introduction

Materials and methods

Chemicals

Semen collection, processing and extender preparation

Semen evaluation

o
Analysis of standard semen parameters

Chlortetracycline (CTC) staining

Malondialdehyde (MDA) concentrations

Flow cytometry

Mitochondrial activity

Phosphatidylserine translocation assay

Statistical analysis
Results

In vitro comparison of egg yolkbased and soybean lecithinbased extenders for cryopreservation of ram
semen Original Research Article
Theriogenology, Volume 73, Issue 4, 1 March 2010,Pages 480-487
M. Forouzanfar, M. Sharafi, S.M. Hosseini, S. Ostadhosseini, M. Hajian, L. Hosseini, P. Abedi, N. Nili, H.R. Rahmani,
M.H. Nasr-Esfahani
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Abstract | Figures/Tables | References

Abstract
Substitution of egg yolk with soybean lecithin may reduce hygienic risks in extenders. Though a few studies have
been performed on the effect of soybean lecithin in bull, to date evaluation of ram semen in vitro fertility after
cryopreservation with use of soybean lecithin has not been studied. This study assessed the effect of 1% or 2%
(wt/vol) soybean lecithin (L1 or L2) or 15% or 20% (vol/vol) egg yolk (E15 or E20) supplemented with 5% or 7%
glycerol (G5 or G7) in a Tris-based medium for cryopreservation of ram (Oviss arries) semen. Although no significant
difference was observed in pattern of capacitation, the best results in terms of sperm motility, viability postthaw, and
cleavage rates were observed with L1G7 (51.9 4.8%, 48.1 3.5%, and 79.6 3.9%, respectively) and E20G7
(51.8 2.9%, 46.7 4.0%, and 72.9 6.4%, respectively). Our results also showed that 1% lecithin and 20% egg
yolk was superior to 2% lecithin and 15% egg yolk. In terms of cleavage rate, 7% glycerol was superior to 5%
glycerol. No significant difference was obtained between groups in terms of blastocysts rate per cleaved embryo.
Therefore, we concluded that the optimal concentration of lecithin and egg yolk is 1% and 20%, respectively, along
with 7% glycerol. In addition, our results suggest that lecithin can be used as a substitute for egg yolk.

Article Outline

1. Introduction

2. Materials and methods

2.1. Extender preparation

2.2. Semen collection and processing

2.3. Evaluation of sperm after freezing-thawing

2.3.1. Sperm motility

2.3.2. Sperm viability

2.3.3. Acrosomal status

2.4. In vitro fertilization

2.4.1. Oocyte in vitro maturation

2.4.2. In vitro fertilization

2.4.3. In vitro culture

2.5. Statistical analysis
3. Results

Seminal plasma proteins and their relationship with sperm motility in Santa Ines
rams
Original Research Article
Pages 94-100
M.A.M. Rodrigues, C.E.A. Souza, J.A.M. Martins, J.P.A. Rego, J.T.A. Oliveira, G. Domont, F.C.S. Nogueira, A.A.
Moura
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Abstract | Figures/Tables | References

Abstract
Recently, comprehensive studies were conducted regarding the reproductive development, age at puberty,
spermatogenesis and the protein profile in the seminal plasma of Santa Ines rams. Despite the abundant information
obtained from these studies regarding these tropically adapted rams in Brazil, it is still unclear how sperm parameters
relate to the expression of molecular components of the reproductive tract. In this regard, the present study was
conducted to determine if sperm parameters were empirically associated with the seminal plasma proteins described
in two-dimensional electrophoresis maps. Seminal plasma proteins were separated by two-dimensional
electrophoresis and the respective maps electronically analyzed. Protein spots associated with higher or lower sperm
motility were then identified by tandem mass spectrometry. Based on sperm motility, the ejaculates were divided into
two groups: those containing up to 80% motile cells (G1; n = 11) and those with more than 80% motile sperm
(G2; n = 10). On average, 236 spots per gel were detected. Eleven spots were significantly more intense in gels from
animals with ejaculates characterized by higher semen motility scores (G2). The intensity of three other spots was
higher in gels from the G1 group. All spots differentially expressed for G1 and G2, were present in at least 90% of the
gels. From the 13 spots differentially expressed in G1 and G2, four spots were identified by tandem mass
spectrometry. Spots expressed with more intensity in the ejaculates with higher sperm motility (G2) were identified as
arylsulfatase A and zinc-alpha-2-glycoprotein. On the other hand, two spots associated with G1 were identified as
ram seminal vesicles protein 22 kDa (RSVP-22) and bodhesin-2. Knowledge of these identities represents a crucial
step toward the comprehension of how specific seminal plasma proteins are related to sperm motility.

Article Outline

1. Introduction

2. Materials and methods

2.1. Semen collection and two-dimensional electrophoresis of the seminal plasma proteins

2.2. Protein identification by MALDI-ToF/ToF mass spectrometry

3. Results

4. Discussion

Analysis of factors contributing to the efficiency of the in vitro production of

transgenic goat embryos (Capra hircus) by handmade cloning (HMC)
Original Research Article
Pages 163-172
A.F. Pereira, C. Feltrin, K.C. Almeida, I.S. Carneiro, S.R.G. Avelar, A.S. Alcntara Neto, F.C. Sousa, C.H.S. Melo,
R.R. Moura, D.I.A. Teixeira, L.R. Bertolini, V.J.F. Freitas, M. Bertolini
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Abstract
Cloning by Somatic Cell Nuclear Transfer (SCNT) still is challenging and inefficient. Recently, the handmade cloning
(HMC) procedures have been successfully applied to livestock species. The aim of this study was to compare the
effect of distinct oocyte sources (in vivo vs. post-mortem) and the final cytoplasmic embryo volume (85% or
2 50%) on fusion rates and on the developmental potential of Day-1 or Day-7 cloned transgenic goat embryos
produced by HMC procedures. Cloned embryos were reconstructed by HMC using skin fibroblast donor cells
established from a transgenic goat. Oocytes were obtained in vivo by laparoscopic oocyte recovery (LOR) from
hormonally stimulated females or post-mortem from slaughterhouse ovaries from nonstimulated goats, resulting in no
differences in the number of aspirated follicles, cumulus-oocyte complexes (COCs), and viable COCs per goat.
However, the COC recovery rate was higher for slaughterhouse ovaries (86.0%) than for LOR (73.0%). Also,
cytoplasmic volume (85% vs. 2 50%) had no effect on fusion rates after embryo reconstruction. Using
slaughterhouse ovaries for cloning, a total of 18.0% (27/150) and 12.7% (19/150) of the in vitro-cultured embryos
developed to the compact morula and blastocyst stages on Day 7. However, no recipients became pregnant on Day
30 following the transfer of Day-1 or Day-7 embryos. In conclusion, the use of slaughterhouse ovaries was as
valuable to supply oocytes for the production of cloned goat embryos by HMC as the in vivo approach. The HMC was
proven a simple alternative for the production of cloned transgenic goat embryos.

Article Outline

1. Introduction

2. Materials and methods

2.1. Animal care and biosafety

2.2. Chemicals, reagents and media

2.3. Experimental design

2.4. Generation and establishment of somatic cell primary cultures

2.4.1. Establishment and expansion of primary cell cultures

2.4.2. Cell growth profile

2.5. Oocyte source and in vitromaturation (IVM) Experiment 1

2.6. In vitro and in vivo development of cloned goat embryos by handmade cloning (HMC)
Experiment 2

2.6.1. Processing of IVM oocytes for preparation of cytoplasts

2.6.2. Donor cell preparation for nuclear transfer

2.6.3. Membrane fusion, chemical activation and in vitro embryo culture

2.6.4. Zona free SCNT-embryo transfer

2.7. Data analyses

3. Results

3.1. In vitro growth pattern of the transgenic donor cells

3.2. Retrieval efficiency of goat cumulus-oocyte complexes obtained in vivo by laparoscopy

(LOR) or post-mortem from slaughterhouse ovaries

3.3. In vitro production of transgenic cloned goat embryos and embryo transfer

4. Discussion

Acknowledgments

Piglets born from handmade cloning, an innovative cloning method without micromanipulation Original

Research Article
Theriogenology, Volume 68, Issue 8, November 2007,Pages 1104-1110
Y. Du, P.M. Kragh, Y. Zhang, J. Li, M. Schmidt, I.B. Bgh, X. Zhang, S. Purup, A.L. Jrgensen, A.M. Pedersen, K.
Villemoes, H. Yang, L. Bolund, G. Vajta
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Abstract | Figures/Tables | References

Abstract
Porcine handmade cloning (HMC), a simplified alternative of micromanipulation based traditional cloning (TC) has
been developed in multiple phases during the past years, but the final evidence of its biological value, births of piglets
was missing. Here we report the first births of healthy piglets after transfer of blastocysts produced by HMC. As a
cumulative effect of technical optimization, 64.3 2.3 (mean S.E.M.) reconstructed embryos from 151.3 4.8
oocytes could be obtained after 34 h manual work, including 1 h pause between fusion and activation. About half
(50.1 2.8%,n = 16) of HMC reconstructed embryos developed to blastocysts with an average cell number of 77 3
(n = 26) after 7 days in vitroculture (IVC). According to our knowledge, this is the highest in vitro developmental rate
after porcine somatic cell nuclear transfer (SCNT). A total of 416 blastocysts from HMC, mixed with 150 blastocysts
from TC using a cell line from a different breed were transferred surgically to nine synchronized recipients. Out of the
four pregnancies (44.4%) two were lost, while two pregnancies went to term and litters of 3 and 10 piglets were
delivered by Caesarean section, with live birth/transferred embryo efficiency of 17.2% (10/58) for HMC. Although
more in vivoexperiments are still needed to further stabilize the system, our data proves that porcine HMC may result
in birth of healthy offspring. Future comparative examinations are required to prove the value of the new technique for
large-scale application.

Article Outline

1. Introduction

2. Materials and methods

2.1. Oocyte collection and in vitro maturation (IVM)

2.2. Handmade cloning (HMC)

2.3. Traditional cloning (TC)

2.4. Embryo culture and transfer

2.5. Fixation and cell number determination of in vitro developed embryos

2.6. Microsatellite analysis

3. Results

3.1. In vitro development of SCNT embryos derived from HMC and TC

3.2. In vivo development of SCNT blastocysts derived from either HMC or TC

4. Discussion

Piglets born from handmade cloning, an innovative cloning method without micromanipulation Original

Research Article
Theriogenology, Volume 68, Issue 8, November 2007,Pages 1104-1110
Y. Du, P.M. Kragh, Y. Zhang, J. Li, M. Schmidt, I.B. Bgh, X. Zhang, S. Purup, A.L. Jrgensen, A.M. Pedersen, K.
Villemoes, H. Yang, L. Bolund, G. Vajta
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Abstract | Figures/Tables | References

Abstract
Porcine handmade cloning (HMC), a simplified alternative of micromanipulation based traditional cloning (TC) has
been developed in multiple phases during the past years, but the final evidence of its biological value, births of piglets
was missing. Here we report the first births of healthy piglets after transfer of blastocysts produced by HMC. As a
cumulative effect of technical optimization, 64.3 2.3 (mean S.E.M.) reconstructed embryos from 151.3 4.8
oocytes could be obtained after 34 h manual work, including 1 h pause between fusion and activation. About half
(50.1 2.8%,n = 16) of HMC reconstructed embryos developed to blastocysts with an average cell number of 77 3
(n = 26) after 7 days in vitroculture (IVC). According to our knowledge, this is the highest in vitro developmental rate
after porcine somatic cell nuclear transfer (SCNT). A total of 416 blastocysts from HMC, mixed with 150 blastocysts
from TC using a cell line from a different breed were transferred surgically to nine synchronized recipients. Out of the
four pregnancies (44.4%) two were lost, while two pregnancies went to term and litters of 3 and 10 piglets were
delivered by Caesarean section, with live birth/transferred embryo efficiency of 17.2% (10/58) for HMC. Although
more in vivoexperiments are still needed to further stabilize the system, our data proves that porcine HMC may result
in birth of healthy offspring. Future comparative examinations are required to prove the value of the new technique for
large-scale application.

Article Outline

1. Introduction

2. Materials and methods

2.1. Oocyte collection and in vitro maturation (IVM)

2.2. Handmade cloning (HMC)

2.3. Traditional cloning (TC)

2.4. Embryo culture and transfer

2.5. Fixation and cell number determination of in vitro developed embryos

2.6. Microsatellite analysis

3. Results

3.1. In vitro development of SCNT embryos derived from HMC and TC

3.2. In vivo development of SCNT blastocysts derived from either HMC or TC

4. Discussion